Covalently reactive transition state analogs and methods of use thereof

ABSTRACT

Improved methods for the production, selection and inhibition of catalytic antibodies are disclosed.

This application is a continuation in part application of U.S.application Ser. No. 09/862,849 filed May 22, 2001, which is divisionalapplication of U.S. application Ser. No. 09/046,373 filed Mar. 23, 1998,now U.S. Pat. No. 6,235,714. This application also claims priority toU.S. Provisional Application 60/280,624 filed Mar. 31, 2001, the entiredisclosure of each of the foregoing applications being incorporated byreference herein.

Pursuant to 35 U.S.C. §202(c) it is acknowledged that the U.S.Government has certain rights in the invention described herein, whichwas made in part with funds from the National Institutes of Health,Grant Numbers: HLA4126, HL59746, AI31268, CA80312, and AI46029.

FIELD OF THE INVENTION

This invention relates to the fields of immunology, molecular biologyand medicine. More specifically, the invention provides novel methodsand compositions for stimulating the production of novel catalyticantibodies and inhibitors thereof. Also provided are improved methodsfor screening phage display libraries expressing catalytic antibodies.

BACKGROUND OF THE INVENTION

Several publications and patent documents are referenced in thisapplication in order to more fully describe the state of the art towhich this invention pertains. The disclosure of each of thesepublications is incorporated by reference herein.

The observation that vasoactive intestinal peptide (VIP) is cleaved byantibodies (Abs) from asthma patients provided early evidence that Absmay possess peptidase activity. This observation has been reproducedindependently by Suzuki et al. Autoantibody catalysis is not restrictedto catalysis of VIP. Autoantibodies in Hashimoto's thyroiditis catalyzethe cleavage of thyroglobulin. Further evidence for autoantibodycatalysis has been provided by reports of DNase activity in Abs fromlupus patients. The bias towards catalytic antibody (Ab) synthesis inautoimmune disease is supported by observations that mouse strains witha genetic predisposition to autoimmune disease produce esterase Abs athigher levels when compared to control mouse strains in response toimmunization with a transition state analog.

Like noncatalytic Abs, peptidase Abs are capable of binding antigens(Ags) with high specificity mediated by contacts at residues from the VLand VH domains. The purified H and L subunits are known to beindependently capable of binding Ags, albeit with lower affinity thanthe parent Ab. X-ray crystallography of Ab-Ag complexes have shown thatthe VL and VH domains are both involved in binding the antigen (Ag). Theprecise contribution of the two V domains varies in individual Ab-Agcomplexes, but the VH domain may contribute at a somewhat greater level,because CDRH3 tends to be longer and more variable in sequence comparedto CDRL3.

The initial complexation of a polypeptide Ag by a peptidase Ab isfollowed by cleavage of one or more peptide bonds. Just prior tocleavage, contacts with the catalytic residues of the antibody areestablished with the peptide bond in the transition state. See FIG. 1.The ability to hydrolyze peptide bonds appears to reside in the VLdomain. This conclusion is based on the cleavage of VIP by polyclonalautoantibody L chains, monoclonal L chains isolated from multiplemyeloma patients and their recombinant VL domains, and recombinant Lchains raised by immunization with VIP. The H chains of polyclonal andmonoclonal Abs to VIP are capable of VIP binding but are devoid of thecatalytic activity. The VH domain can nevertheless influence thepeptidase activity by “remote control”, because in binding to VIP remotefrom the cleavage site, it can influence the conformation of the bindingsite as shown by the peptidase activity of F, constructs composed of thecatalytic anti-VIP VL domain linked to its VH domain. The anti-VIP VHdomain exerted beneficial effects and an irrelevant VH domain exerteddetrimental effects on the catalytic activity, as evaluated by thevalues of VIP binding affinity and catalytic efficiency. The proposedexistence of distinct catalytic and antigen binding subsites incatalytic Abs is consistent with data that Abs generally contain largecombining sites, capable of accommodating 15-22 amino acids ofpolypeptide substrates, and that substrate regions distant from thecleavage site are recognized by the Abs. Thus, the VH domain offers ameans to control the specificity of the catalytic site.

The present invention provides novel compositions and methods forstimulating production of catalytic antibodies and fragments thereof.Catalytic antibodies with specificity for target antigens provide avaluable therapeutic tool for clinical use. Provided herein are improvedmethods for identifying, isolating and refining catalytic antibodies forthe treatment of a variety of medical diseases and disorders, includingbut not limited to infectious, autoimmune and neoplastic disease. Suchcatalytic antibodies will also have applications in the fields ofveterinary medicine, industrial and clinical research and dermatology.

SUMMARY OF THE INVENTION

According to one aspect of the invention, methods and compositions areprovided herein for stimulating catalytic antibody production topredetermined target antigens, including but not limited to thoseinvolved in pathogenic and neoplastic processes. Covalently reactivetransition state analogs (CRTSAs) are described which stimulate theproduction of catalytic antibodies with therapeutic value in thetreatment of a variety of medical conditions, including autoimmunitydisorders, microbial diseases, lymphoproliferative disorders and cancer.The catalytic antibodies of the invention may also be usedprophylatically to prevent certain medical disorders, including but notlimited to septic shock, systemic inflammatory disease and acuterespiratory distress syndrome.

The covalently reactive transition state analogs, (CRTSAs) of thepresent invention contain three essential elements and have thefollowing formula: R₁-E-R₂ wherein R₁ is a peptide sequence of anepitope of a target protein antigen, E is an electrophilic covalentlyreactive center bearing a partial or full negative charge and R₂ is anelectron withdrawing or electron donating substituent, R2 optionallyfurther comprising a flanking peptide sequence. The CRTSAs of theinvention optionally comprise Y which is a basic residue (Arg or Lys oran analog thereof) at the P1 position (first amino acid on theN-terminal side of the reaction center), Y may also comprise an electronwithdrawing or electron donating substituent as shown in FIGS. 16 and17.

In one aspect of the invention, CRTSAs are administered to a livingorganism under conditions whereby the CRTSAs stimulate production ofspecific catalytic antibodies. The catalytic antibodies are thenpurified. Antibodies so purified are then adminstered to a patient inneed of such treatment in an amount sufficient to inactivate antigensassociated with a predetermined medical disorder.

According to another aspect of the present invention, a method isprovided for treating a pathological condition related to the presenceof endogenously expressed catalytic antibodies. Examples of suchabnormal pathological conditions are certain autoimmune disorders aswell as lymphoproliferative disorders. The method comprisesadministering to a patient having such a pathological condition apharmaceutical preparation comprising covalently reactive transitionstate analog capable of irreversibly binding the endogenously producedcatalytic antibodies, in an amount sufficient to inhibit the activity ofthe antibodies, thereby alleviating the pathological condition. In thisembodiment, the CRTSA contains a minimal B epitope only to minimize theimmunogenicity of the CRTSA.

According to another aspect of this invention, a pharmaceuticalpreparation is provided for treating a pathological condition related tothe presence of endogenously produced catalytic antibodies. Thispharmaceutical preparation comprises a CRTSA in a biologicallycompatible medium. Endogenously produced catalytic antibodies areirreversibly bound and inactivated upon exposure to the CRTSA. Thepreparation is administered an amount sufficient to inhibit the activityof the catalytic antibodies.

In another aspect of the invention, methods for passively immunizing apatient with a catalytic antibody preparation are provided. Catalyticantibodies are infused into the patient which act to inactivate targeteddisease associated antigens.

In an alternative embodiment, should the patient experience unwantedside effects, the activity of the infused catalytic antibodies may beirreversibly inactiviated by administering the immunizing CRTSA to saidpatient. Again, the immunogenicity of the CRTSA in this embodiment wouldbe reduced via the inclusion of a minimally immunogenic B cell epitope.A T cell universal epitope would be omitted in this CRTSA.

In yet another aspect of the invention, active immunization of patientsis achieved by administering the CRTSAs of the invention in aCRTSA-adjuvant complex to a patient to be immunized. At least 2subsequent booster injections of the CRTSA-adjuvant complex at 4 weekintervals will also be adminstered. Following this procedure, thepatient sera will be assessed for the presence of prophylactic catalyticantibodies.

A further aspect of the invention comprises methods for screening phageor B cells for expression of catalytic antibodies. In this embodiment,phage or B cells are screened with a CRTSA and those phage or B cellwhich bind the CRTSA are isolated and characterized further. Methods forisolating and cloning the DNA encoding catalytic antibodies from phageor B cells so isolated are also within the scope of the presentinvention.

Finally, catalytic sFv and light chains are also with encompassed withinthe present invention.

The methods and CRTSAs of the present invention provide notableadvantages over currently available compounds and methods forstimulating catalytic antibodies specific for predetermined targetantigens. Accordingly, the disclosed compounds and methods of theinvention provide valuable clinical reagents for the treatment ofdisease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A free energy diagram for antibody catalysis involvingstabilization of the substrate ground state (ΔG_(s)) and transitionstate (ΔG_(TS)). ΔG⁺ _(uncat) and ΔG⁺ _(cat) correspond to activationenergies for the uncatalyzed and catalyzed reactions, respectively. Kmis a function of the extent of ground state stabilization (ΔG_(s)).Kcat/Km is a function of the extent of transition state stabilizationrelative to the catalyst-substrate ground state complex.

FIG. 2. Compounds 1-5.

FIG. 3. Binding of monoester 3 and diester 5 by trypsin.Streptavidin-peroxidase stained blots of SDS-gels showing trypsin boundto 3 without and with preincubation with DFP (lanes 1 and 2,respectively) and to 5 without and with preincubation with DFP (lanes 3and 4, respectively). Pretreatment of trypsin (1 μM) with DFP (1 mM) orsolvent was for 30 min followed by incubation with 3 (200 M) or 5 (20M), gel filtration, precipitation of the effluent at the void volumewith trichloroacetic acid, dissolution of the pellets in 2% SDS, boiling(5 min) and electrophoresis on SDS-gels.

FIG. 4. Identification of monoester-binding site in trypsin by massspectrometry. (A) Following treatment of trypsin with 4, the adductswere subjected to tryptic digestion and affinity chromatography onimmobilized avidin. The molecular ion at m/z 3738 corresponds to trypsinresidues 189-218 derivatized by 4. Also detected were underivatizedpeptide 189-218 (m/z 3186) and the phosphonic acid derivative from 4(m/z 569), presumably formed by partial decomposition of thephosphonylated fragment during sample preparation. Signals at m/z 3221and 2003 are avidin fragments (see text for explanation). (B) Proposedstructure of 4-derivatized trypsin fragment. C represents theS-carbamoylmethylated cysteine residue.

FIG. 5. Kinetics of trypsin inactivation by monoester 2 (A) and diester1(B). Reaction initiated by mixing trypsin [1 mM (2) or 0.1 mM (1)] withvarying inhibitor concentrations [2 0.4 (●), 0.6 (▪), 0.8 (▴), 1.0 (◯),1.2 (□) mM; 10.5 (●), 1.0 (▪), 1.5 (▴), 2.0 (◯), 2.5 (□) mM] at 37° C.Aliquots withdrawn at various time points (2, 5-20 min; 1, 0.5-5 min),diluted 500-fold (2) or 50-fold (1), substrate (EAR-MCA, 0.2 mM) addedand residual enzyme activity measured fluorometrically. The standarddeviation of the residuals (sy.x) for linear regression fits were nevermore than 0.07 (A) and 0.23 (B).

FIG. 6. Irreversible inhibition of (A) thrombin and (B, C) YZ17 activityby monoester 3. (A)●: Thrombin (4 unit/ml) preincubated with 1000×3concentrations shown on X-axis (37° C., 30 min) followed by 1000×dilution in buffer containing substrate (VPR-MCA, 25 M) and incubationfor 3 h. ◯: Thrombin (0.004 unit/ml) incubated with 3 and 25 M substratewithout the preincubation step. Data are means of 3 closely agreeingreplicates (s.d. <12.7%). Thrombin activity without 3 25.5-27.9 FU/h (3independent experiments). Background fluorescence 21 FU. (B) YZ17 (20nM) incubated with 3 and 0.4 mM substrate without the preincubationstep. Arrow: final concentration of 3 in panel C. (C) YZ17 (200 nM)preincubated for varying periods (X-axis) with 0.5 mM 3 followed by 100×dilution in solvent containing substrate (EAR-MCA, 0.4 mM) yielding 3concentration shown by the arrow in panel B. Catalytic activity wasmeasured over 16 h. Data are means of 3 closely agreeing replicates(s.d. <8.3%). YZ17 activity without 3, 16.0-16.9 FU/h. Backgroundfluorescence 19.3 FU.

FIG. 7. Binding of monoester 4 by Fv and L chain clones.Streptavidin-peroxidase stained blots of SDS-gels showing reactionmixtures of monoester 4 (500 μM) treated for 24 h with metal affinitypurified recombinant Ab fragments. Clone Fv YZ17 (2 μM) without (lane 1)and with DFP pretreatment (5 mM, lane 2); L chain clone SK-35 (4.4 μM,lane 3); and L chain clone c23.5 (2 gM, lane 4). Lane 5, molecularweight markers. Sample processing for electrophoresis was as in FIG. 3.

FIG. 8. DFP and phosphonate esters.

FIG. 9. DFP and diester II inhibition of VIPase antibody fragments.Inhibition of 11 clones by DFP and 6 clones by II was analyzed. (▴)germline L chain c23.5, (♥) somatically mature L clone c23.5, (▪) Lchain hk14, (♦) Fv mRT3, (●) L chains U2, U19, U15, U7, U30, U4, U16listed in order of decreasing residual activity in the presence of DFP.L chains U4 and U16 were studied for II inhibition. Substrate about 50μM (Tyr ₁₀₋₁₂₅ I)VIP (25 K c.p.m.). Inhibitors: DFP 1 mM; II 0.1 mM.Reaction conditions 3 hours, 37° C. Catalyst concentration 2-50 nM[adjusted to give about 40% (Tyr ₁₀₋₁₂₅ I)VIP hydrolysis]. Values (meansof closely agreeing duplicates) are percent of hydrolysis observed incontrol assays without inhibitor.

FIG. 10. Irreversible inhibition of peptidase activity by DFP anddiester II: effect of substrate. L chain clone U16 (1.3 μM) was treatedthe inhibitors (1 mM DFP, 0.1 mM II) or assay diluent in the presence orabsence of VIP (1 μM, 30 min, 37° C.). Excess inhibitor and VIP wereremoved by gel-filtration and the protein fraction assayed for (Tyr₁₀₋₁₂₅ I)VIP as in FIG. 2 (U16 concentration, 33 nM).

FIG. 11. Diester II binding by proteolytic Ab fragments. (A) Gelfiltration of II-Fv mRT3 complexes. Fv (1.3 μM) was treated with II (100μM; 30 min) and the mixture (200 μl) subjected to chromatography on aSuperose-12 column. Aliquots of column fractions (100 μl) were analyzedin duplicate for II-Fv complexes by ELISA (see Methods). Inset,silver-stained SDS-polyacrylamide gel (8-25%) of fraction 30,corresponding to the Fv peak. (B). Streptavidin-peroxidase stained blotsof SDS-polyacrylamide gels showing II-adducts of Fv mRT3 (lane 1), Lchain c23.5 (lane 2), L chain hk14 (lane 3) and trypsin (lane 4). Lane5, 6 and 7 are silver-stained Fv mRT3, L chain c23.5 and L chain hk14.Treatment of Fv mRT3 (0.4 μM), c23.5 L chain (1.8 μM), hk14 L chain (0.4μM) and trypsin (0.2 μM) with II (20 μM) was for 30 min, 37° C. IIstaining of monomer Ab fragments is evident. Dimeric L chain c23.5 (55kD) and trypsin breakdown products display low level staining. (C).Streptavidin-peroxidase stained dot blot of L chain c23.5 (1.8 μM)treated with II (1 μM, 2 hours, 25° C.) in the absence (dot 1) andpresence of 80, 10, 1 and 0.1 μM VIP (dots 2, 3, 4 and 5, respectively).Dot 6, 2 μM bovine serum albumin treated with II. Dot 7, background IIstaining in the absence of protein.

FIG. 12. Enriched catalytic activity in phage Abs selected on diester IIand monoester III. Pro-Phe-Arg-MCA cleavage by unselected andII-selected lupus L chains (1 μM in A, black columns; 1 μM in B) andlupus III-selected Fv populations (0.2 μM in A, hatched columns).Substrate 400 μM. Elution of II-bound L chain phages was with 2-PAM (20mM, 24h, 25° C.) and of III-bound Fv and L chain phages, with2-mercaptoethanol. Catalysis assays were carried out using IMAC purifiedsoluble Ab fragments. Values are means of 3 replicates after subtractionof background peptide-MCA cleaving activity in extracts from bacteriaharboring phagemid DNA without an Ab insert (about 1 FU/hour).

FIG. 13. Substrate selectivity of chemically selected Abs fragments. Lchain GG63 (bottom). Fv YZ17 (top). Catalyst, 80 nM; peptide-MCAsubstrates, 200 μM. Cleavage rates were computed from duplicate assaysby linear regression from plots of fluorescence intensity vs reactiontime.

FIG. 14. Inhibition of monoester III-selected Fv YZ17 by diester IV.Substrate EAR-MCA, 200 μM; Fv YZ17, 20 nM. Incubation for 15 hours.Substrate hydrolysis in the absence of inhibitor was 194 FU (100%).Inhibitors were preincubated at 1.2-fold the indicated concentration for1 h with Fv prior to substrate addition. Values are means of 2replicates.

FIG. 15. Monoester III and diester IV binding by chemically selectedantibody fragments Streptavidin-peroxidase stained blots ofSDS-electrophoresis gels showing purified Fv YZ17 complexed to IV andIII (lanes 1 and 2, respectively); purified L chain GG63 complexed to IVand III (lanes 3 and 4, respectively); periplasmic extracts of Fv cloneYZ17 and control cells harboring phagemid vector without an antibodyinsert stained with diester II (lane 5 and 6, respectively). Lane 7,silver-stained SDS-gel of purified Fv clone YZ17; lane 8, anti-c-mycstained blot thereof. Treatment of the purified proteins (1 μM) andperiplasmic extracts (dialyzed against assay diluent) with II (10 μM),III (100 μM) and IV (10 μM) was for 30 min, 37° C.

FIG. 16. Representative CRTSA structures are shown.

FIG. 17. Representative CRTSA structures are shown.

FIG. 18. Neutral diesters and monoesters suitable for use in the methodsof the invention are shown.

FIGS. 19A and 19B. A list of antigens targeted by conventionalmonoclonal antibodies showing clinical promise. Such antigens aresuitable targets for the catalytic antibodies of the present invention.

FIG. 20. An exemplary CRTSA of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Methods are disclosed for stimulating synthesis of catalytic antibodiesof predetermined specificity by the immune system. In one embodiment ofthe invention compositions and methods are provided for the generationof catalytic antibodies to a peptide antigen of choice. In anotherembodiment, compositions and methods are provided which are useful inpassive immunotherapy modalities for the treatment of cancer and othermedical conditions.

In another embodiment of the invention, vaccination protocols aredescribed which elicit catalytic Ab production to predetermined viral orpathogenic antigens. The covalently reactive transition state antigenanalogs disclosed preferentially stimulate the production of catalyticantibodies. Such antibodies provide superior protection againstinfection due to the presence of catalytic action against the targetantigen which results in its permanent inactivation. Additionally, asingle catalytic Ab molecule may be reused to inactivate multipleantigen molecules as compared to noncatalytic Abs which bind antigenreversibly and stoichiometrically.

The CRTSA of the invention is composed of certain basic elements. Theseinclude an electrophilic reaction center, and at least one peptidesequence corresponding to an epitope in a target antigen. In a preferredembodiment, the electrophilic reaction center is selected from the groupof molecules shown in FIGS. 16 and 17. The CRTSA of the invention canoptionally comprise a positively charged amino acid residue adjacent tothe electrophilic reaction center and a second peptide sequence whichtogether the first flank the electrophilic reaction center.

Immunization with transition state analogs (TSAs) has been proposed as ameans to derive Abs that can bind the transition state, and thus lowerthe activation energy barrier for the reaction. The commonly usedphosphonate analogs contain a tetrahedral phosphorus atom and anegatively charged oxygen atom attached to the phosphorus. Formation ofthe transition state of peptide bond cleavage is thought to involveconversion of the trigonal carbon atom at the cleavage site to thetetrahedral state, and acquisition of a negative charge by the oxygen ofthe carbonyl group. The conventional phosphonate TSAs may induce,therefore, the synthesis of Abs capable of binding the oxyanionstructure and the tetrahedral configuration of the transition state.However, Abs to these TSAs, while capable of accelerating comparativelyundemanding acyl transfer reactions, cannot effectively catalyze peptidebond cleavage. An antibody to a phosphinate TSA has recently beenreported to slowly cleave a stable primary amide. It is possible thatthe anti-phosphinate Ab may permit superior transfer of a proton to theamide nitrogen at the scissile bond, compared to the more commonanti-phosphonate Abs, which might account for its better catalyticactivity.

Most enzymologists hold that phosphonate TSAs fail to elicit efficientcatalytic Abs because they are poor transition state mimics, and becausemultiple transition states are involved. Enzymes use activated aminoacid sidechains to catalyze peptide bond cleavage. For instance, the Serhydroxyl group acquires enhanced nucelophilicity and the capability tomediate covalent catalysis due to formation of an intramolecular,hydrogen bonded network of the Ser, His and Asp residues. Thephosphonate analogs do not contain structural elements necessary to bindthe nucleophilic reaction center. Induction of the covalent catalysiscapability in Abs is therefore unattainable using conventionalphosphonate TSAs. Further, these TSAs do not exploit the existence ofthe germline encoded, serine protease site in Abs.

Covalently reactive antigen analogs (CRAA) have been described in U.S.Pat. No. 6,235,714, the entire disclosure of which is incorporated byreference herein.

Electrophilic CRTSAs are capable of reacting with the nucleophilicserine residue of the catalytic Abs. These antigen analogs have beenapplied to select catalysts from the antibody libraries. The logicalextension of this strategy is to force the utilization of the serineprotease sites for the synthesis of antibodies specific for individualtarget antigens, such as the epidermal growth factor receptor (EGFR).This can be achieved by immunization with the aforementionedelectrophilic CRTSAs. Such CRTSAs promote clonal selection of B cellsexpressing the germline encoded serine protease sites on their cellsurface. Further, the specificity for EGFR, for example, can be ensuredby incorporating an appropriate antigenic epitope from EGFR which willflank the covalently reactive antigen analog structure.

Catalytic Ab synthesis has been documented in autoimmune disease [2, 4].Further, the immune system is capable of producing Abs that catalyze thecleavage of exogenous antigens, including the cleavage of MV proteingp120. However, patients infected with the virus do not mount acatalytic Ab response to gp120. The HIV CRTSAs discussed herein willforce the immune system to synthesize protective catalytic antibodies toHIV. Data are presented herein which support this approach. gp120 hasbeen selected as the target antigen for the following reasons: (a) It isan essential constituent of HIV-1 for productive infection of hostcells; (b) As a virus-surface protein, gp120 is readily accessible toAbs; and (c) Certain anti-gp120 Abs have been shown to arrest HIVinfection.

Prior art methods have described combined administration of both CRAAand TSA separately. The CRTSA of the invention combine both of thesereactivities in a single molecule. Incorporating all of the features ofthe transition state in a single analog molecule has unique advantagesthat are not expressed in analogs known in the art. Simultaneousrecognition of these features by the catalysts is essential to ensurethat the analog-catalyst binding occurs at the highest possibleaffinity. Catalysts that bind the CRTSAs can thus be anticipated tostabilize the corresponding transition state more strongly than thecatalysts that bind TSAs alone or CRAAs alone. There is no assurancethat a CRAA binding catalyst will also bind the TSA, or that a TSAbinding catalyst will bind the CRAA. Thus, phage or B cell selectionprotocols in which sequential binding to TSAs and CRAAs are employedwill not yield the same catalysts as those that bind the CRTSAs. Similarconsiderations apply in response to induction of antibody catalystsynthesis in response to immunization with these analogs. Binding of theanalog to Ig on the cell surface stimulates clonal proliferation, duringwhich the antibody variable regions undergo random sequencediversification. Cells expressing mutants with the highest affinity forthe analog possess a survival advantage, in that they can preferentiallybindg the analog and undergo further rounds of cell division. Sequentialimmunization with the CRAA and the TSA (heterologous immunization) mayprovide for selection of mutants that can bind both analogs, butstrengthened binding of one analog may be accompanied by weaked bindingfor the other analog, as there is no selective advantage for the cell toretain its original Ig specificity. Thus, the use of CRTSAs asimmunogens can be anticipated to induce the synthesis of catalysts thatstabilize the transition state more strongly than antibodies producedfollowing sequential immunization with CRAAs and TSAs.

Phosphonate monoesters have been assumed to serve as noncovalenttransition state analogs for enzymes capable of catalyzingtransacylation reactions. Here, we present evidence for the covalentreaction of certain serine proteinases and peptidase antibody fragmentswith monophenyl amino(4-amidinophenyl)methanephosphonate derivatives.Stable adducts of the N-biotinylated monophenyl ester with trypsin andantibody fragments were evident under conditions that disruptnoncovalent interactions. The reaction was inhibited by the activesite-directed reagent diisopropyl fluorophosphate. Mass spectrometry ofthe fragments from monoester-labeled trypsin indicated phosphonylationof the active site. Irreversible inhibition of trypsin- andthrombin-catalyzed hydrolysis of model substrates was observed. Kineticanalysis of inactivation of trypsin by the N-benzyloxycarbonylatedmonoester suggested that the first-order rate constant for formation ofcovalent monoester adducts is comparable to that of the diester adducts(0.47 min⁻¹ vs. 2.0 min⁻¹). These observations suggest that the covalentreactivity of phosphonate monoesters contributes to their interactionswith serine proteinases, including certain proteolytic antibodies.

In yet another embodiment of the invention, the reactivity ofphosphonate ester probes with several available proteolytic antibody(Ab) fragments was characterized. Irreversible, active-site directedinhibition of the peptidase activity was evident. Stable phosphonatediester-Ab adducts were resolved by column chromatography and denaturingelectrophoresis. Biotinylated phosphonate esters were applied forchemical capture of phage particles displaying Fv and light chainrepertoires. Selected Ab fragments displayed enriched catalytic activityinhibitable by the selection reagent. Somewhat unexpectedly, aphosphonate monoester also formed stable adducts with the Abs. Improvedcatalytic activity of phage Abs selected by monoester binding was alsoevident. Turnover values (k_(cat)) for a selected Fv construct and alight chain against their preferred model peptide substrates were,respectively, 0.5 min⁻¹ and 0.2 min⁻¹, and correspondingMichaelis-Menten constants (Km) were, 10 μM and 8 μM. The covalentreactivity of Abs with phosphonate esters suggests their ability torecapitulate the catalytic mechanism utilized by classical serineproteases.

The CRTSAs of the invention and the resulting catalytic antibodies haveat least three major applications. The first application is directed tothe generation of catalytic antibodies in either humans or animalsfollowing immunization with a CRTSA designed for a particular medicaldisorder. The catalytic antibodies so generated would then beadministered to patients to inactivate targeted antigen moieties. Inthis scenario, should the patient experience adverse side effects, theimmunizing CRTSA may be administered to irreversibly inactivate thecatalytic antibody. The CRTSAs in this embodiment would be synthesizedwith a B cell epitope only in order to minimize immunogenicity.

In the second application, CRTSAs may be administered to patients forthe purposes of actively immunizing the patient against particularpathological to generate a state of protective immunity. These CRTSAswould be administered as a CRAA-adjuvant complex.

Finally, the CRTSAs of the invention may be administered to patients whoare currently expressing catalytic antibodies in association with amedical disorder such as autoimmune disease or multiple myeloma. CRTSAsmay be designed with specifically react with the antibodies present.Inhibition of catalytic function should result in an amelioration of thedisease state. Again, these CRTSAs are designed to contain a minimallyimmunogenic B cell epitope only.

The detailed description set forth below describes preferred methods forpracticing the present invention. Methods for selecting and preparingCRTSAs, stimulating the production of catalytic antibodies topredetermined antigens and improved methods for isolating the same aredescribed.

I. Selection and Preparation of CRTSAs

The covalently reactive transition state antigen analogs of theinvention are prepared using conventional organic synthetic schemes. Thenovel CRTSAs of the invention contain an electron withdrawing orelectron donating substituent flanked by at least one peptide sequencederived from proteins associated with a particular peptide antigen to betargeted for cleavage and the intended use of the CRTSA.

Selection of suitable flanking amino acid sequences depends on theparticular peptide antigen targeted for cleavage. For example, viralcoat proteins, certain cytokines, and tumor-associated antigens containmany different epitopes. Many of these have been mapped usingconventional monoclonal Ab-based methods. This knowledge facilitates thedesign of efficacious covalently reactive transition state antigenanalogs useful as catalytic antibody inhibitors as well as inducers ofcatalytic antibodies with catalytic activities against predeterminedtarget antigens.

The amino acids flanking the reaction center represent the sequence ofthe targeted epitope in defined polypeptides that play a role indisease, or to which autoantibodies are made in disease.

The structural features of the CRTSAs are intended to permit specificand covalent binding to immature, germline encoded antibodies as well asmature antibodies specialized to recognize the targeted epitope. Basedon the tenets of the clonal selection theory, the CRTSAs are alsointended to recruit the germline genes encoding the catalytic antibodiesfor the synthesis of mature antibodies directed towards the targetedepitope.

Polypeptides to be targeted include soluble ligands and the membranebound receptors for these ligands.

Microbial proteins can also be targeted for catalysis by the antibodiesof the present invention. These include but are not limited to gp120,gp160, Lex 1 repressor, gag, pol, hepatitis B surface antigen, bacterialexotoxins (diptheria toxin, C. tetani toxin, C. botulinum toxin,pertussis toxin).

Neoplastic antigens will also be incorporated into therapeuticallybeneficial CRTSAs. These include but are not limited to EGF, TGFα, p53products, prostate specific antigen, carcinoembryonic antigen,prolactin, human chorionic gonadotropin, c-myc, c-fos, c-jun,p-glycoproteins, multidrug resistance associated proteins,metalloproteinases and angiogenesis factors.

Receptors for neoplastic antigens will also be targeted forantibody-mediated catalysis. These include EGFR, EGFR mutants, HER-2,prolactin receptors, and steroid receptors.

Inflammatory mediators are also suitable targets for catalysis.Exemplary molecules in this group include TNF, IL-1 beta, IL-4 as wellas their cognate receptors.

Preexisting catalytic antibodies are found in autoimmune disease andlymphoproliferative disorders. The harmful actions of these catalyticantibodies will be inhibited by administering CRTSAs to patients. CRTSAsdesigned to be weakly immunogenic will be administered which covalentlyinteract with antibody subunits with specificity for VIP,Arg-vasopressin, thyroglobulin, thyroid peroxidase, IL-1, IL-2,interferons, proteinase-3, glutamate decarboxylase.

For maximum selectivity, the flanking peptide sequences comprise anepitope which is targeted for cleavage. For example, an epitope presentin the epidermal growth factor receptor is incorporated in a CRTSA ofthe present invention. In another embodiment of the invention, anepitope present in HIV gp120 is incorporated into a CRTSA.

The discussion below relates to an exemplary CRTSA for the treatment ofHIV infection which comprises both a B cell epitope and a T cell epitopeto maximize the immunogenicity of the CRTSA. While HIV specific CRTSAare described one of ordinary skill in the art appreciates that thefollowing concepts and methodology may be applied to design CRTSAimmunologically specific for any target epitope. CRTSAs of the B cellepitope will be designed to elicit catalytic Abs. An exemplary B cellepitope is derived from the CD4 binding site, which is generallyconserved in different HIV-1 strains. The CD4 binding site of gp120 is asuitable target, further, because unlike many other epitopes, it isaccessible to Abs on the native viral surface. It is known that the CD4binding site is a conformational determinant.

In the present invention, preparation of a catalytic Ab that recognizesa specific portion of the CD4 binding site (as opposed to the entire CD4binding site) is described. Additional peptide epitopes in gp120 (orother HIV proteins) that might be suitable targets for catalytic Abswill also be identified. Because cleavage of gp120 may lead to globalchanges in the protein conformation, accompanied by loss of biologicalactivity, certain gp120 peptide epitopes may be appropriate targets ofcatalytic Abs even if they do not participate directly in HIV-1 bindingto host cells or HIV-1 interactions with intracellular components. Theseand other targets are also contemplated to be within the scope of thepresent invention.

T cell help for Ab synthesis is potentially subject to restriction indifferent individuals due to major histocompatibility complex (MHC)polymorphism. In the present invention, mouse strains with well-definedgenetic backgrounds will be used as models for the elicitation ofcatalytic immunity in response to B-T epitope conjugates. A “universal”T-helper epitope recognized promiscuously by various MHC class IIalleles will be utilized. Another benefit of this approach is that it isreadily adaptable to human clinical trials.

The envelope glycoproteins of HIV-1 are synthesized as a single 160 kDprecursor, gp160. This protein is cleaved at the Arg511-Ala512 bond by acellular protease, producing gp120 and the integral membrane proteingp41. The biological activity of gp120 is a key ingredient in initialbinding of host cells by HIV-1, propagation of the virus, and its toxiceffects on uninfected neurons and other cells. Binding of aconformational epitope of gp120 to CD4 receptors on host cells is thefirst step in HIV-1 infection. Individual amino acids constituting thisepitope appear to be located in the second (C2), third (C3), and fourth(C4) conserved gp120 segments. These are gp120 residues 256, 257,368-370, 421-427 and 457. Monoclonal antibodies that bind the CD4binding site have been described. Since the CD4 binding site is aconformational epitope, distant residues that are not themselvesconstituents of the epitope may be important in maintaining the abilityto bind CD4.

gp120 interactions with other host cell proteins are also essential forvirus propagation. For example, binding of gp120 by calmodulin may beinvolved in HIV-1 infectivity, as revealed by the inhibitory effect ofcalmodulin antagonists. Asp180 located between the V1 and V2 regions ofgp120 is critical for viral replication. Similarly, the V3 loop may beessential for infectivity. It is clear, therefore, that structuraldeterminants in gp120 other than those constituting the CD4 binding siteare necessary for virus genome replication, coat protein synthesis, andvirus particle packaging.

Trypsinization of gp120 blocks its neurotoxic effects. Treatment ofHIV-1 particles with trypsin, mast cell tryptase or Factor Xa attenuatestheir infectivity. Cleavage of gp120 at residues 269-270 or 432-433destroys CD4 binding capability, whereas cleavage at residues 64-65,144-145, 166-167, 172-173 or 315-316 does not affect CD4 binding. On theother hand, cleavage at the Arg315-Ala316 peptide bond located in the V3loop of gp120 by a cellular protease is believed to be essential forproductive viral infection. A dipeptidylpeptidase expressed on the hostcell-surface (CD26) has been proposed as being responsible for cleavageat Arg315-Ala316. This cleavage site is located in the principalneutralizing determinant (PND), which is a component of the V3 gp120loop to which protective Abs are readily synthesized. It has beenhypothesized that Ab binding to the PND blocks the cleavage of gp120 bya host cell protease, resulting in HIV neutralization. There is noevidence that the PND plays a direct role in HIV binding by CD4, but itsparticipation in binding by the HIV coreceptors has been suggested.

Efficient Ab synthesis by B cells is dependent in part on recruitment ofT helper cells, which, once sensitized, secrete the necessarystimulatory cytokines and activate B cells by direct contact mediatedthrough accessory molecules, such as CD4 on T helper cells and B7 on Bcells. Recruitment of Ag-specific T cells occurs through recognition bythe T cell receptor (TCR) of the complex of a processed Ag epitope boundto MHC class II molecules.

The peptide-based vaccines are formulated by covalently linking a T cellepitope to a B cell epitope, against which the host synthesizes Abs. TheT epitope binds MHC class II molecules on the surface ofantigen-presenting cells, and the MHC class II complex of the B-Tepitopes is then bound by the TCR. Different individuals in an outbredspecies express different MHC class II alleles involved in Agpresentation to T cells (I-E and I-K loci). Ideally, a peptide vaccineshould be free of MHC restrictions, i.e., a robust Ab response should beprovoked regardless of the MHC class II variations involved in Agpresentation.

The interactions between MHC class II molecules, the TCR and the Agepitope are quite promiscuous. Thus, certain peptides can serve asuniversal T epitopes, i.e., these peptides can bind the different MHCclass II alleles efficiently. Further, there is no apparent restrictionof recognition of the peptides at the level of the different types ofTCRs. Such peptides are suitable T epitope components in vaccinesdesigned to neutralize HIV through elicitation of a protective Abresponse, as described in the present invention.

By incorporating appropriate structure in the immunogens capable ofinducing the synthesis of Abs that combine specificity for gp120 withrapid peptide bond cleaving activity, an immunotherapeutic agent for thetreatment of AIDS will be generated.

Methods are provided for the synthesis of peptide analog formulationsthat elicit the synthesis of specific and efficient catalytic Abscapable of protecting against HIV infection. Earlier studies havesuggested that polyreactive catalytic activity of germline encoded Abscan be recruited and improved by immunization of mice with theserine-reactive CRTSA of a gp120 peptide. The elicitation of a catalyticAb response should provide superior protection against HIV-1 infectioncompared to a noncatalytic Ab response.

The following synthetic immunogens will be prepared and assessed:

-   -   A) synthetic immunogens        -   a) the phosphonate monoester CRTSA of a B cell epitope of            gp120 (residues 421-436) conjugated to a T-helper epitope            from tetanus toxoid (residues 830-844) [designated B-T            epitope];        -   (b) the unmodified peptide form of the B-T epitope.    -   (B) Immunize non-autoimmune mice (strain B10.BR) and autoimmune        mice (MRL/1 pr) with the two immunogens from (A) and study the        following activities of IgG purified from the sera:        -   (a) binding and cleavage of the CRTSA B-T epitope, and the            unmodified B-T epitope;        -   (b) binding and cleavage of monomer full-length gp120;            and (c) binding and cleavage of native cell-surface-bound            gp120.            Immunogens

The prototype vaccine capable of eliciting catalytic antibodies to HIVcontains: 1) an epitope to which B cells can make high affinityantibodies (B epitope); 2) an epitope that is bound by MHC class IIantigens and presented to T cells (T epitope); and 3) a structural mimicof the transition state formed during peptide bond cleavage, which isintended to provoke the synthesis of antibodies capable of stabilizingthe transition state, and thus catalyzing the cleavage reaction.

B epitope component: Loss of infectivity following cleavage of gp120 canbe achieved by directing the catalyst to cleave a peptide bond locatedin an epitope of gp120 that plays an important role in the infectionprocess. Note that cleavage of gp120 at a bond distant from thebiologically important determinants may also lead to loss of gp120function, because the conformation of the gp120 fragments may be altered“globally” relative to the parent protein. The probability ofneutralizing viral infectivity can be increased by directing the Ab torecognize an epitope that is a known target of neutralizing Abs.Cleavage of the CD4 binding site is an attractive mechanism to achieveHIV neutralization for the following reasons: CD4-gp120 binding is anessential step in HIV entry into host cells; cleavage of the CD4 bindingat the 432-433 bond by trypsin is known to block the ability of gp120 tobind CD4; Abs to the CD4 binding site are known to inhibit HIVinfection; the CD4 binding site on native gp120 expressed on the HIVsurface is exposed to the environment (as opposed to several otherepitopes of monomeric gp120 that are buried in native gp120 oligomers)[32]; and, the CD4 binding site is quite conserved in different subtypesof HIV-1. The linear peptide sequence composed of gp120 residues 421-436has been selected as the B epitope component of the immunogen in thepresent project (KQIINMWQEVGKAMYA (SEQ ID NO: 1)). Mutagenesis studieshave shown that this region of gp120 make important contributions in CD4binding.

T epitope component: To recruit T cell help for synthesis of anti-gp120Abs, a fifteen amino acid peptide (QYIKANSKFIGITEL (SEQ ID NO: 2)corresponding to residues 830-844 of tetanus toxin will be placed on theN terminal side of the B epitope. The presence of the T epitope in thevaccine construct eliminates the need to conjugate the B epitope to alarge carrier protein. Several previous studies have shown thatcomparatively short linear peptides that include a T and a B epitope arecapable of provoking efficient Ab synthesis to the B epitope [42]. Thetetanus toxin T epitope to be employed in the present invention is knownto serve as a T epitope in hosts expressing diverse class II alleles,and has been characterized, therefore, as a “universal” T epitope [43].Further, a gp120 B epitope linked to this T epitope is described toinduce anti-gp120 Ab synthesis. The “universality” of the T epitope,although deduced from human studies, probably extends to the mouse,because class II restrictions tend to be conserved phylogenetically.Regardless of the possible differences on this point between man andmouse, the mouse strains to be utilized in the present invention havebeen matched for class II alleles involved in recruitment of T cell helpfor Ab synthesis (A^(k)E^(k) haplotype), eliminating concern thatdifferential T helper recruitment might contribute to variations incatalytic Ab responses.

Assembly of immunogens: Synthesis of the 31 residue ground state B-Tconstruct (designated unmodified B-T epitope) composed of tetanus toxinresidues 830-844 at the N terminus and gp120 residues 421-436 at the Cterminus will be done by conventional solid phase synthesis on anApplied Biosystems synthesizer. Mass spectrometry and ¹H and ¹³CNMR willbe done to confirm the structures.

II. Administration of CRTSAs

CRTSAs as described herein are generally administered to a patient as apharmaceutical preparation. The term “patient” as used herein refers tohuman or animal subjects.

The pharmaceutical preparation comprising the CRTSAs of the inventionare conveniently formulated for administration with a acceptable mediumsuch as water, buffered saline, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol and the like), dimethylsulfoxide (DMSO), oils, detergents, suspending agents or suitablemixtures thereof. The concentration of CRTSAs in the chosen medium willdepend on the hydrophobic or hydrophilic nature of the medium, as wellas the other properties of the CRTSA. Solubility limits may be easilydetermined by one skilled in the art.

As used herein, “biologically acceptable medium” includes any and allsolvents, dispersion media and the like which may be appropriate for thedesired route of administration of the pharmaceutical preparation, asexemplified in the preceding paragraph. The use of such media forpharmaceutically active substances is known in the art. Except insofaras any conventional media or agent is incompatible with the CRTSA to beadministered, its use in the pharmaceutical preparation is contemplated.

Conventional immunization methods will applied to induce catalytic Absynthesis. Three intraperitoneal and one intravenous injections of theimmunogens (about 100 μg peptide each) will be administered. The finalimmunization will be carried out intravenously. RIBI will be used in theanimal studies. For human use, alum will be employed as the adjuvant.Alum is approved for human use and has previously been shown to provokeAb synthesis to a B-T epitope similar to those proposed in the presentinvention. RIBI is a low toxicity replacement for Freund's CompleteAdjuvant, and reproducibly facilitates good Ab responses to a variety ofAgs. Analysis of two adjuvants is advantageous because the quality andmagnitude of Ab responses to vaccines can be influenced by adjuvants,via effects of the cytokines and TH subpopulations recruited by theadjuvants on B.

CRTSAs may be administered parenterally by intravenous injection intothe blood stream, or by subcutaneous, intramuscular or intraperitonealinjection. Pharmaceutical preparations for parenteral injection arecommonly known in the art. If parenteral injection is selected as amethod for administering the molecules of the invention, steps must betaken to ensure that sufficient amounts of the molecules reach theirtarget cells to exert a biological effect.

The pharmaceutical preparation is formulated in dosage unit form forease of administration and uniformity of dosage. Dosage unit form, asused herein, refers to a physically discrete unit of the pharmaceuticalpreparation appropriate for the patient undergoing treatment. Eachdosage should contain a quantity of active ingredient calculated toproduce the desired effect in association with the selectedpharmaceutical carrier. Procedures for determining the appropriatedosage unit are well known to those skilled in the art.

The pharmaceutical preparation comprising the CRTSA may be administeredat appropriate intervals, for example, twice a day until thepathological symptoms are reduced or alleviated, after which the dosagemay be reduced to a maintenance level. The appropriate interval in aparticular case would normally depend on the condition and thepathogenic state sought to be treated in the patient.

III. Administration of Catalytic Antibodies

The catalytic antibodies described herein are generally administered toa patient as a pharmaceutical preparation.

The pharmaceutical preparation comprising the catalytic antibodies ofthe invention are conveniently formulated for administration with aacceptable medium such as water, buffered saline, ethanol, polyol (forexample, glycerol, propylene glycol, liquid polyethylene glycol and thelike), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents orsuitable mixtures thereof. The concentration of catalytic antibodies inthe chosen medium will depend on the hydrophobic or hydrophilic natureof the medium, as well as the other properties of the catalyticantibodies. Solubility limits may be easily determined by one skilled inthe art.

As used herein, “biologically acceptable medium” includes any and allsolvents, dispersion media and the like which may be appropriate for thedesired route of administration of the pharmaceutical preparation, asexemplified in the preceding paragraph. The use of such media forpharmaceutically active substances is known in the art. Except insofaras any conventional media or agent is incompatible with the catalyticantibody to be administered, its use in the pharmaceutical preparationis contemplated.

Conventional passive immunization methods will be employed whenadministering the catalytic antibodies of the invention. In a preferredembodiment, Abs will be infused intravenously into the patient. Fortreatment of certain medical disorders, steps must be taken to ensurethat sufficient amounts of the molecules reach their target cells toexert a biological effect.

The lipophilicity of the molecules, or the pharmaceutical preparation inwhich they are delivered may have to be increased so that the moleculescan arrive at their target locations. Furthermore, the catalyticantibodies of the invention may have to be delivered in a cell-targetedcarrier so that sufficient numbers of molecules will reach the targetcells. Methods for increasing the lipophilicity and targeting oftherapeutic molecules, which include capsulation of the catalyticantibodies of the invention into antibody studded liposomes, are knownin the art.

The catalytic antibodies that are the subject of the present inventioncan be used as antibody fragments or whole antibodies or they can beincorporated into a recombinant molecule or conjugated to a carrier suchas

polyethylene glycol. In addition any such fragments or whole antibodiescan be bound to carriers capable of causing the transfer of saidantibodies or fragments across cell membranes as mentioned above.Carriers of this type include but are not limited to those described(Cruikshank et al. in the Journal of Acquired Immune DeficiencySyndromes and Human Retrovirology, March 1997).

The pharmaceutical preparation is formulated in dosage unit form forease of administration and uniformity of dosage. Dosage unit form, asused herein, refers to a physically discrete unit of the pharmaceuticalpreparation appropriate for the patient undergoing treatment. Eachdosage should contain a quantity of active ingredient calculated toproduce the desired effect in association with the selectedpharmaceutical carrier. Procedures for determining the appropriatedosage unit are well known to those skilled in the art. For example, thehalf-life of syngeneic IgG in the human is about 20 days. Over thisperiod, 60,480 Ag molecules will be cleaved by one molecule of anantibody with a turnover of 2.1/min (which is the turnover of a humananti-VIP L chain isolated from a phage display library [14]. It can beseen, therefore, that the peptidase antibodies can express considerablymore potent antigen neutralizing activity than stoichiometric,reversibly-binding molecules. Note that the antibody light chainsdiscussed here were selected based on their antigen-binding affinity, aprocedure that favors tight binding to the antigen, but will not selectcatalysts with the best turnover. Antibodies produced by immunizationwith CRTSAs and isolated by appropriate selection methods, as disclosedhere, will express considerably greater turnover. Such catalyticantibodies can be used to treat disease at substantially lower doses ofcorresponding noncatalytic antibodies.

The pharmaceutical preparation comprising the catalytic antibodies maybe administered at appropriate intervals, for example, twice a weekuntil the pathological symptoms are reduced or alleviated, after whichthe dosage may be reduced to a maintenance level. The appropriateinterval in a particular case would normally depend on the condition andthe pathogenic state sought to be treated in the patient.

CRTSAs will be selected that will generate catalytic antibodies forpassive or active immunotherapy that will fulfill the standard criteriafor acceptable prophylatic or therapeutic agents: (1) Cleavage of thetarget peptide antigen by the catalytic antibody will lead to abeneficial change in a pathological process by either functionallyactivating or functionally inactivating the target peptide antigen; and(2) Administation of said catalytic antibodies or the induction of theirproduction in the body by means of immunization with a CRTSA will resultin a favorable therapeutic index such that the clinical benefit gainedoutweighs the morbidity associated with andy side-effects. Discussionsof how such criteria are established for the acceptability ofprophylatic or therapeutic agents are common in the art can can be foundin such texts as Guide to Clinical Trials by Bert Spilker, Raven Press,New York, 1991.

Suitable categories of prophylatic or therapeutic target peptideantigens for the practice of the present invention include but are notlimited to cytokines, growth factors, cytokine and growth factorreceptors, proteins involved in the transduction of stimuli intiated bygrowth factor receptors, clotting factors, integrins, antigen receptors,enzymes, transcriptional regulators particularly those involved incellular program (differentiation, proliferation and programmed celldeath) control, other inducers of these cellular programs, cellularpumps capable of expelling anticancer agents, microbial and viralpeptide antigens.

Conventional monoclonal antibodies that act to inhibit the function ofparticular target molecules are among the most common type oftherapeutic agent under development for clinical use by biotechnologyand pharmaceutical companies. Some of these have shown substantialclincal promise and any exposed peptide target antigens that are part ofthe same molecular functional unit are therefore shown to beparticularly well suited as potential targets for the catalyticantibodies that are the subject of the present invention. The catalyticantibodies comtmplated in the present invention will constitute a majorinprovement over such conventional monoclonals becaule of their abilityto affect many target molecules vs. just one and because of theresulting dramatic decrease in the cost of treatment. The availabilityof peptide bonds within these targeted antigens can be determined bymethods well established in the art including but not limited to ademonstration of cleavage following exposure to proteolytic enzymes andcatalytic light chains capable of cleaving a range of peptide bonds.

A listing of some of the antigens targeted by conventional monoclonalantibodies showing clinical promise and the corresponding medicalindications are shown in FIGS. 19A and 19B.

Thus, it is an object of the present invention to provide a covalentlyreactive transition state antigen analog, and a method of producing it,which is capable of 1) provoking the generation of catalytic andnucleophilic antibodies specific to a predetermined antigen of theinvention and/or 2) irreversibly inhibiting the catalysis by antibodiesassociated with autoimmune disease and certain lymphoproliferativedisorders. Further objects reside in providing processes for preparingantigens and their corresponding antibodies, and in providing assays andmethods of using these antibodies as beneficial therapeutic agents.

The following examples are provided to facilitate an understanding ofthe present invention. They are not intended to limit the invention inany way.

EXAMPLE 1 Irreversible Inactivation of Trypsin by a PhosphonateMonoester Derivative

Phosphonate monoesters are comparatively stable compounds thought toapproximate the stereoelectronic features of the rate-limitingtransition state of certain transacylation reactions (TAs²).Consequently, they are useful in study of catalytic mechanisms,particularly for ceratin Abs capable of catalyzing esterolytic reactions(1,2). TA binding by enzyme active sites is usually viewed as involvingnoncovalent interactions at the tetrahedral phosphorus atom and itsoxyanion, corresponding to the tetrahedral carbon atom and thedeveloping charge on the carbonyl group in the transition state (3,4,and references therein). Recently, phosphonate monoester derivativeswere observed to bind irreversibly to certain naturally occurringproteolytic Abs (5), suggesting that noncovalent mimicry of theoxyanionic transition state may not fully explain the reactivity ofthese compounds. Previously, the monoesters have been held inert tonucleophilic attack by serine proteinase active sites owing to thedelocalized negative charge carried by the oxygen atoms. Phosphonatediesters, on the other hand, are well known to bind the active site ofserine proteinases covalently by phosphonylation of the hydroxylfunction of active site serine residue (6).

The catalytic mechanisms utilized by naturally occurring Abs areunderstood only minimally. In the present study, therefore, we employeda better-characterized catalyst to study the covalent reactivity of thephosphonate monoesters, i.e., trypsin. Phosphonate monoesters were boundcovalently by the active site of trypsin, the catalytic activity wasinhibited irreversibly, and the rate constant for formation of themonoester-enzyme adduct was comparable to that of the homologousdiester. Studies using thrombin and recombinant Ab fragments confirmedthe irreversible binding and inhibition of catalytic activity,suggesting that the covalent reactivity is a general feature ofphosphonate monoester interaction with serine proteinases.

Materials and Methods

(2-Biotinamido)ethylamido-3,3′-dithiodipropionic acidN-hydroxysuccinimide ester, succinimidyl 6-(biotinamide)hexanoate,trypsin (porcine, type IX), diisopropyl fluorophosphate (DFP), andVPR-MCA were from Sigma (St. Louis, Mo.); acetonitrile (HPLC grade) wasfrom Fisher Scientific (Pittsburgh, Pa.); EAR-MCA was from PeptidesInternational (Louisville, Ky.); 8-25% PHAST electrophoresis gels, ECLmolecular mass markers, streptavidin-horseradish peroxidase conjugate,and ECL kits were from Amersham Pharmacia Biotech (Piscataway, N.J.);and N-tosyl-phenylalanine chloromethyl ketone-treated bovine trypsin wasfrom Pierce (Rockford, Ill.). 1-Methyl-2-pyrrolidinone,N,N-dimethylformamide and N,N-diisopropylethylamine were of peptidesynthesis grade (Applied Biosystems, Foster City, Calif.). Otherreagents for chemical synthesis were of reagent grade. Thrombin (human,a) was purified as described in (7). HPLC was conducted using a WatersDELTA-600 system equipped with a 2487 UV/VIS detector (Milford, Mass.)and YMC-pack ODS AM columns [YMC USA (Milford, Ma.); 4.6.times.250 mm(for analysis) and 20.times.250 mm (for purification)] with thefollowing mobile phase: A, 0.05% trifluoroacetic acid in water; B, 0.05%trifluoroacetic acid in acetonitrile [flow rate 1.0 ml/min (foranalysis) or 10 ml/min (for purification)]. Purity of the compoundsobtained is represented by % peak area of the desired compound in HPLCchromatograms at 220 nm. ¹H-NMR (400 MHz) spectra were measured inD.sub.20-acetonitrile-d3 containing 3-(trimethylsilyl)-1-propanesulfonicacid sodium salt as internal standard. Stock solutions of compounds 1-5(10 mM) in N,N-dimethylformamide were stored at −80° C. and wereanalyzed by HPLC periodically to confirm their integrity.

Synthesis of Phosphonate Compounds 1-5

Diphenyl N-(benzyloxycarbonyl)amino(4-amidinophenyl)methanephosphonate(1). This compound was synthesized according to (8) with slightmodifications. The iminoester prepared from diphenyl[N-(benzyloxycarbonyl)amino](4-cyanophenyl)methane phosphonate (1.5 g,3.0 mmol) was dissolved in a mixture of methanol (30 ml) and 0.5 M NH₃in 1,4-dioxane (15 ml), the solution stirred at room temperatureovernight, solvent was removed and the oily residue dissolved in CHCl₃(30 ml). Diethyl ether (100 ml) was added, the precipitate collected byfiltration, washed with diethyl ether (30 ml×5) and dried in vacuo[yield 1.2 g (73%)]. The crude product was judged sufficiently pure forbiotinylation. For use in kinetic studies, further purification wascarried out by HPLC (100 mg crude 1) yielding 36 mg 1. [t_(R) 31.58 min,purity 99% (A:B 80:20 to 20:80 in 60 min); m/z (ESI) 516 (M+H)⁺, 1031(2M+H)⁺].

Monophenyl N-(benzyloxycarbonyl)amino(4-amidinophenyl)methanephosphonate(2). Crude 1 (200 mg) was allowed to hydrolyze in acetonitrile (5 ml)and 2.5% aq. Na₂CO₃ (15 ml) at room temperature overnight, pH wasadjusted to 3 with 6 N HCl, and the mixture subjected to preparativeHPLC and lyophilization, yielding a colorless powder [yield 83 mg; t_(R)17.70 min, purity 91% (A:B 80:20 to 20:80 in 60 min); m/z (ESI) 440(M+H)⁺, 880 (2M+H)⁺].

Monophenyl-N-[2-(biotinamido)ethylamido-3,3′-dithiodipropionyl]amino(4-amidinophenyl)methanephosphonate(3). 2 (83 mg, 0.19 mmol) was dissolved in 30% HBr in acetic acid (5 ml)and the resulting yellow solution was stirred at room temperature for 3h. Procedures described for 1 were carried out to precipitate, wash anddry the amine derivative. The resulting anine and(2-biotinamido)ethylamido-3,3′-dithiodipropionic acidN-hydroxysuccinimide ester (100 mg, 0.17 mmol) were allowed to react in1-methyl-2-pyrrolidinone (4 ml) containing N,N-diisopropylethylamine (88ml, 0.56 mmol) overnight at room temperature. Preparative HPLC andlyophilization yielded a colorless fluffy powder [yield 110 mg (83%);t_(R) 18.11 min, purity 94% (A:B 90:10 to 20:80 in 45 min); m/z (ESI)766 (M+H)⁺; ¹H-NMR spectrum consistent with assigned structure].

MonophenylN-[6-(biotinamido)hexanoyl]amino(4-amidinophenyl)methanephosphonate (4).The benzyloxycarbonyl group of 2 was removed and the resulting amine (50mg, 0.09 mmol) was biotinylated by means of succinimidyl6-(biotinamide)hexanoate (49 mg, 0.11 mmol) as described for 3. Thecrude product was purified by HPLC to give a colorless fluffy powder[yield 51 mg (89%); t_(R) 15.05 min, purity >99% (A:B 90:10 to 20:80 in45 min); m/z (ESI) 645 (M+H)⁺].

Diphenyl-N-[2-(biotinamido)ethylamido-3,3′-dithiodipropionyl]amino(4-amidinophenyl)methanephosphonate(5). The benzyloxycarbonyl group of crude 1 was removed and biotin wasincorporated in the resulting amine (110 mg, 0.18 mmol) by means of(2-biotinamido)ethylamido-3,3′-dithiodipropionic acidN-hydroxysuccinimide ester (125 mg, 0.22 mmol) as described for 3. Thecrude product was purified by HPLC to give a colorless fluffy powder[yield 20 mg (13%); t_(R) 24.95 min, purity 96% (A:B 90:10 to 20:80 in45 min); m/z (ESI) 843 (M+H)⁺, 865 (M+Na)⁺; ¹H-NMR spectrum consistentwith assigned structure].

Binding and Enzyme Acivity Assays

Binding assay. Trypsin and Ab fragment clones were allowed to react withphosphonates 3 and 4 under conditions described in legends for FIGS. 3and 7. The reaction mixtures were subjected to gel filtration,precipitated with an equal volume of 20% (w/w) trichloroacetic acid, thepellet was collected by centrifugation, SDS was added to 2%, the samplesboiled in a water bath for 5 min and analyzed by SDS-PAGE on 8-25% PHASTgels. The gels were electroblotted onto nitrocellulose membrane(TransBlot; Biorad, Hercules, Calif.), blocked with 5% nonfat milk inPBS-T, washed with PBS-T, treated with a streptavidin-horseradishperoxidase conjugate (1:1000) in 10 mM sodium phosphate, pH 7.4,containing 137 mM NaCl, 2.7 mM KCl, and 0.025% Tween-20 (PBS-T) andenzyme-bound phosphonate detected using an ECL kit and X-OMAT film(Kodak).

Inactivation assay. Trypsin (0.75 nM) in PBS-T was incubated in thepresence of 3 (12.5 M-2.5 mM) at 37° C. for 30 min in 96-well MicrofluorII white plates (Dynex Technologies, Va.). EAR-MCA was added to thesolutions (concentrations of trypsin, substrate and 3 in the assaymedium were 0.6 nM, 0.2 mM and 10 M-2.0 mM, respectively) and theresulting coumarin derivative after 1 h incubation was determined byfluorometry (l_(em) 470 nm, l_(ex) 360 nm; LS50B luminescencespectrometer equipped with a plate reader, Perkin Elmer, Shelton,Colo.). To assess irreversibility, phosphonate compounds in the reactionmixtures were removed by gel-filtration in PBS-T (Spin-out 6000 columns;Chemicon International, Temecula, Calif.) or diluted to noninhibitoryconcentrations prior to measurement of catalytic activity. For kineticanalyses, aliquots of the trypsin-phosphonate reaction mixtures werewithdrawn at various intervals and diluted 500-fold (1) or 50-fold (2)with PBS-T. EAR-MCA (0.4 mM, 25 l) was added to 25 l of the dilutedreaction mixtures, and the residual activity was measuredfluorometrically. Dissociation constants for the initial noncovalentcomplex (K_(i)) and the rate constants for the conversion of thenoncovalent complex to the irreversibly inactivated enzyme (k₂) weredetermined from Kitz-Wilson's plots (9), in which the reciprocalapparent first-order inactivation rate constant (k_(app)) was plottedversus reciprocal inhibitor concentration. Thrombin and Ab catalysisassays were carried out similarly using VPR-MCA (25 mM) and EAR-MCA (0.4mM), respectively. Preparation of recombinant Ab fragments and theirsources have been described in (5,10).

Mass Spectrometry

Trypsin (1 mg) was allowed to react with 4 (3 mg) in 1 ml 100 mM NH₄HCO₃(pH 8.0) at room temperature. Unreacted 4 was removed by gel filtration(10DG column, Bio-Rad). The protein-containing fraction (2 ml) wastreated with urea (8 M) and dithiothreitol (20 mM, 22° C., 30 min),followed by iodoacetamide (50 mM, 22° C., 30 min), and then desalted byHPLC (Vydac protein C4 column; 0.1% trifluoroacetic acid in 5%acetonitrile/water, 5 min, then 0.1% trifluoroacetic acid in 59%acetonitrile/water, 10 min, 1.0 ml/min). The protein fraction wasconcentrated to 950 μl by vacuum centrifugation, the pH adjusted to 8.0using 2 M Tris base, and then treated with N-tosyl-phenylalaninechloromethyl ketone-treated bovine trypsin (50 μg) at room temperatureovernight. To separate biotinylated peptides, the solution was allowedto bind SoftLink Avidin Resin in 10 mM sodium phosphate, pH 7.4,containing 137 mM NaCl, and 2.7 mM KCl (1 ml gel; Promega, Madison,Wis.; 30 min with rotation). The slurry was packed in a polypropylenecolumn and washed with the same buffer (10 ml), and bound peptideseluted with 8 M urea in 100 mM ammonium bicarbonate, pH 8.0, containing10 mM methylamine. The eluate was desalted, lyophilized, and subjectedto MALDI-TOF MS with a-cyano-4-hydroxycinnamic acid as the matrix.

Results

Syntheses of phosphonates 1-5. The structure of phosphonates 2-5 (FIG.2) is based on diphenylN-benzyloxycarbamido(4-amidinophenyl)methanephosphonate (1), which haspreviously been described to be a potent and irreversible inhibitor oftrypsin, thrombin and certain other serine-proteinases (8).N-Benzyloxycarbonylated diester 1 was synthesized essentially accordingto the reported procedure (8), and its monoester derivative 2 wasprepared by hydrolysis of 1. The protecting group N-benzyloxycarbonylwas removed from diphenyl phosphonate 1 and its monophenyl derivative 2,and biotin was introduced at the amino function to yield diester 5 andmonoester 3, respectively. This permitted sensitive detection ofenzyme-phosphonate adducts on electrophoresis gels by anelectrochemiluminescence reaction using a streptavidin-horseradishperoxidase conjugate. Monoester 4 was prepared by essentially the samemethod as for 3 (the two compounds are identical except that 4 containsa non-reducible linker to allow mapping of the trypsin binding siteunder reducing conditions).

Active site-directed covalent binding. Trypsin incubated with 3 and 5was subjected to gel filtration, precipitation with trichloroacetic acidand then boiled in SDS prior to electrophoresis to remove unbound andnon-covalently bound phosphonate. Trypsin bands labeled with monoester 3and diester 5 were observed using a biotin-specificelectrochemiluminescence reaction (FIG. 3). Pretreatment with 1 mM DFP,a classical active site-directed inhibitor of serine proteinases,completely inhibited labeling of the enzyme by 3 and 5, indicating thatboth phosphonate esters bind the enzyme active site.

Direct evidence for active site modification by the phosphonatemonoester was obtained by MS analysis of fragments obtained by trypticdigestion of trypsin adduct followed by affinity chromatography onimmobilized avidin monomers. The molecular ion of the phosphonylatedfragment 189-218 (m/z 3738) was evident (FIG. 4A). This fragmentcontains the active site Ser195 residue. The proposed structure of theadduct (FIG. 4B) is consistent with the observed m/z and is identical tothe “aged” complex formed by phosphonate diesters with a serineproteinase (11,12). Additional constituents identified were: (a)fragment ions corresponding to the dephosphonylated peptide and the freephosphonic acid derivative of 4 (m/z 3186 and 569, respectively); and(b) avidin fragments at m/z 3221 and 2003 corresponding to residues101-128 and 112-128, respectively. As the samples analyzed by MS werepurified by affinity chromatography on immobilized avidin, coincidentalcontamination with the free phosphonic acid derivative (m/z 569) andunreacted peptide (m/z 3186) is unlikely. Presumably, these constituentswere detected because of partial degradation of the phosphonylatedpeptide adduct during sample handling. Avidin fragments detected areattributable to tryptic digestion of the affinity column matrix (thetryptic digest was applied to the avidin column without inactivation ofthe enzyme).

Irreversible inactivation. The reactions of an enzyme (E) with acovalent active site modifier (I) can be represented by the followingscheme:

-   -   where K_(i) is the dissociation constant for the noncovalent        complex (E-I), k₂, the first-order rate constant for formation        of the covalent complex Ei-I, and k₃, the first-order rate        constant for decomposition of Ei-I.

Concentration-dependent inhibition of trypsin-catalyzed EAR-MCAhydrolysis by monoester 3 was observed [9.9-80% inhibition at 0.01-2.0mM 3; s.d. <5.6%, n=3; enzyme 0.6 nM; substrate 0.2 mM; enzyme activitywithout 3,301 FU/h]. The inhibitory effect was irreversible, as judgedby the following observations. Trypsin (200 nM) was pretreated with 3(1.0 mM) for 30 min followed by dilution of the reaction mixture tonon-inhibitory concentrations of 3 prior to assay for enzyme activity(1000-fold dilution; 3 concentration during enzyme assay 1.0 mM). Markedinhibition of trypsin activity was observed (85% inhibition; activitywithout 3, 92-105 FU/h; no inhibition by 1.0 mM 3 was observed unlessthe enzyme was pretreated with the compound prior to incubation withsubstrate). Evidently, the dilution step failed to dissociate theenzyme-monoester complexes formed during the pretreatment step,suggesting an apparent irreversible inhibition mode. Removal of unboundphosphonates by gel filtration of trypsin pretreated with monoester 3(800 mM) and diester 5 (80 mM) at 37° C. (22 h) did not restore theenzyme activity [activity levels following gel filtration of 3-treatedand 5-treated trypsin: 27.6±2.6 FU/h and 5.0±0.1 FU/h, respectively,compared 33.2±2.6 FU/h and 6.5±0.2 FU/h without removal of 3 and 5,respectively; activity levels in control trypsin without phosphonate:288.4±8.4 FU/h (without gel filtration) and 308.2±13.4 FU/h (t=0following gel filtration)]. Moreover, incubation of the 3-enzyme adductisolated by gel filtration for 17 h in buffer failed to restore theactivity appreciably (50.2±7.0 FU/h), suggesting that the Ei-I complexdoes not decompose to yield active enzyme (k₃˜0). Under theseconditions, Kitz-Wilson plots (9) of inhibitor concentration vs apparentfirst-order inactivation rate constants (k_(app)) can be applied fordetermination of K_(i) and k₂.

Kinetic studies were carried out using diester 1 and monoester 2containing N-benzyloxycarbonyl in place of the biotin substituent.Diester 1 has previously been described to inhibit trypsin (8), andpreliminary kinetic analysis in the present study indicated that 1 is asuperior inhibitor of trypsin compared to the biotin-containing diester5 utilized for catalyst binding studies (k_(app)/[I] 3703 and 0.23 M⁻¹sec⁻¹, respectively; determined at 1.5 mM 1 and 0.3 mM 5). Apparently,inclusion of the biotin group interferes with the enzyme-phosphonateinteractions. k_(app) values for monoester 2 and diester 1 were obtainedas the slope of plots of In[V_(t)/V₀] vs time at varying phosphonateconcentration, where V₀ represents initial velocity of substratehydrolysis in the absence of phosphonate and V_(t), initial velocityafter treatment with phosphonate esters (FIG. 5). k₂ for formation ofthe covalent monoester adduct was only 4-fold lower than the diesteradduct (0.47 min⁻¹ vs. 2.0 min⁻¹). K_(i) values for the monoester 2 anddiester 1 noncovalent complexes were 5.2 mM and 7.2 M, respectively.

Irreversible inactivation and binding of thrombin and Ab fragments.Procedures identical to those used for trypsin were applied to studyeffect of monoester on thrombin and a catalytic Ab fragment (clone YZ17,GenBank accession number 12957377; FIG. 6). Pretreatment of thecatalysts with 3 produced inhibition of activity despite dilution of thereaction to non-inhibitory concentrations. Ab clones isolated byselection of phage libraries on a phosphonate monoester (Fv clone YZ17)and a diester (L chain clone SK35; GenBank accession number AF425258)were analyzed for monoester 4 binding. Biotin-containing 27 kD adductsstable to denaturing treatments were observed (boiling, trichloroaceticacid-precipitation, SDS treatment; FIG. 7). Consistent with the tendencyof L chains to form aggregates, the dimer and higher order 4-labeledstates of SK35 L chain were observed. Under equivalent conditions, an Abfragment identified as a catalyst based on random screening assays (10)failed to form stable 4-adduct [L chain clone c23.5 (lane 4); GenBankaccession numbers 896288]. The adduct formation was inhibited by DFP(FIG. 7, lane 2), suggesting that the binding is active site directed.

Phosphonate monophenyl esters 24 were observed to irreversibly bind andinhibit the active site of trypsin. Irreversible interactions ofmonoester 4 with thrombin and a proteolytic Fv clone were also evident.The first-order rate constant for formation of the covalent trypsinadduct of monoester 2 (k₂) is comparable to the corresponding value fordiester 1 adduct. This is noteworthy because the delocalized negativecharge carried by the oxygen atoms is anticipated to result in reducedelectrophilicity. For instance, spontaneous hydrolysis of thephosphonate monoesters under basic conditions occurs 2-3 orders ofmagnitude slower than of the diesters³ (13). Moreover, trypsininactivation by 2 occurs rapidly (k₂/K_(i)=90 M⁻¹ min⁻¹) compared tospontaneous hydrolysis of analogous monoaryl esters⁴ (these reactionscorrespond to dephenoxylation by the enzyme and dephenoxylation by OH⁻,respectively; 13-15). Thus, the inactivation of trypsin by monoester 2may be accelerated by the enzyme itself, as reported to be the case withother organophosphorus inhibitors (16,17). Increased electrophilicreactivity of the phosphorus atom can be anticipated, for instance, ifthe enzyme active site disrupts resonance hybridization around the O—P—Ocenter, resulting in a greater localization of the negative charge onone of the oxygens. An analogous activation scheme is proposed for thecovalent reaction of phosphonate monoesters with -lactamase (18).

K_(i) values computed from Kitz-Wilson plots indicate that the overallstrength of noncovalent enzyme-2 interactions is weaker than forenzyme-1 interactions (ΔG 3.2 and 7.3 kcal/mol at 37° C., respectively;ΔG=−RTInK_(i), where ΔG, R and T represent the Gibbs free energy change,gas constant and absolute temperature, respectively). Interpreting thelow affinity of monoester 2 for trypsin, however, is complicated by thefollowing factors. First, the binding strength derived from K_(i) valuespotentially includes contacts at subsites in the ground states as wellas those unique for the transition state (e.g., oxyanion stabilization).Second, in the case of the diester, stabilizing hydrophobic interactionsat the second phenyl group can not be excluded in view of reportsdescribing preferential recognition of hydrophobic moieties by the S₁′subsite of trypsin (19,20). The K_(i) values, therefore, do not shedlight on the energetic contribution of phosphonate oxyanion interactionswith trypsin. Previous studies have provided strong support for the roleof the phosphonate oxyanion as a mimic of the negative charge found onthe carbonyl oxygen in the transition state of transacylation reactions(21). Despite the comparatively large K_(i) value, phosphonatemonoesters hold promise as analogs capable of combining noncovalent andcovalent reaction pathways to probe serine proteinase active sites. Infuture studies, the available means to improve inhibitory potencyinclude optimization of contacts in the transition state as well as theground state of the enzyme-inhibitor complex, for instance, byincorporation of peptidic groups on the flanks of the phosphonate groupand removal of steric conflicts that may interfere with oxyanionbinding.

With respect to proteolytic Abs, distinct levels of monoester binding bydifferent L chain clones prepared under identical conditions wereobserved, and one L chain clone did not show detectable covalentbinding, judged by denaturing electrophoresis. The apparent presence ofAb nucleophiles with varying levels of monoester 4 binding activity isconsistent with the sequence diversity of Ab combining sites. Theidentity of the nucleophiles in different clones, the structural factorsgoverning their chemical reactivity and the relationship of thephosphonate binding sites with catalysis remain to be elucidated.Moreover, the catalytic activities need not be directly correlated withthe level of covalent binding, as catalysis requires additionalhydrolysis and product release steps. Previous studies have focused onthe link between noncovalent binding of phosphonate monoesters and theesterolytic activity of certain Abs (22). No explanation has beenavailable for the unusual properties of several esterolytic Abs raisedby immunization with the monoesters, including unusual kinetic behaviorinconsistent with simple oxyanion stabilization⁵ (23), apparentirreversible binding to the substrate (24), and the presence of a serineproteinase-like Ser-His catalytic dyad in the Ab combining site (25).Recognition of phosphonate monoester covalent reactivity may helpexplain the discrepancies, as the immunization procedure can behypothesized to allow adaptive maturation of nucleophilic Abs.

REFERENCES FOR EXAMPLE I

-   1. Tramontano, A., Janda, K. D., and Lerner, R. A. (1986) Science    234, 1366-1570.-   2. Pollack, S. J., Jacobs, J. W. and Schultz, P. G. (1986) Science    234, 1570-1573.-   3. Hasserodt, J. (1999) Synlett, 2007-2022.-   4. Golineli-Pimpaneau, B. (2000) Curr. Opin. Struct. Biol. 10,    697-708.-   5. Paul, S., Tramontano, A., Gololobov, G., Zhou, Y.-X., Taguchi,    H., Karle, S., Nishiyama, Y., Planque, S., and George, S. (2001) J.    Biol. Chem. 276, 28314-28320.-   6. Oleksyszyn, J. and Powers, J. C. (1994) in Methods in Enzymology,    Vol. 244. Proteolytic Enzymes: Serine and Cysteine Peptidases    (Barrett, A. J. ed.) pp. 423-441, Academic Press, New York.-   7. Fenton, J. W., II, Fasco, M. J. and Stackrow, A. B. (1977) J.    Biol. Chem 252, 3587-3598.-   8. Oleksyszyn, J., Boduszek, B., Kam, C.-M. and    Powers, J. C. (1994) J. Med. Chem. 37, 226-231.-   9. Kitz, R. and Wilson, I. B. (1962) J. Biol. Chem. 237, 3245-3249.-   10. Gao, Q. S., Sun, M., Rees, A. R. and Paul, S. (1995) J. Mol.    Biol. 253, 658-664.-   11. Bertrand, J. A., Oleksyszyn, J., Kam, C.-M., Boduszek, B.,    Presnell, S., Plaskon, R. R., Suddath, F. L., Powers, J. C. and    Williams, L. D. (1996) Biochemistry 35, 3147-3155.-   12. Bone, R., Sampson, N. S., Bartlett, P. A., Agard, D. A. (1991)    Biochemistry 30, 2263-2272.-   13. Behrman, E. J., Biallas, M. J., Brass, H. J., Edwards, J. O.,    and Isaks, M. (1970) J. Org. Chem. 35, 3063-3075.-   14. Whithey, R. J. (1969) Can. J. Chem. 47, 4383-4387.-   15. Moss, R. A. and Ragunathan, K. G. (1999) Langmuir 15, 107-110.-   16. Schowen, R. L. (1978) in Transition States of Biochemical    Processes (Gandour, R. D., Schowen, R. L. eds.) pp.86, Plenum, New    York.-   17. Kovach, I. M., Larson, M., and Schowen, R. L. (1986) J. Am.    Chem. Soc. 108, 5490-5495.-   18. Pratt, R. F. (1989) Science 246, 917-919.-   19. Schellenberger, V., Turck, C. W., Hedstrom, L., and    Rutter, W. J. (1993) Biochemistry 32, 4349-4353.-   20. Schellenberger, V., Turck, C. W. and Rutter, W. J. (1994)    Biochemistry 33, 4251-4257.-   21. Gigant, B., Charbonnier, J.-B., Eshhar, Z., Green, B. S. and    Knossow, M. (1997) Proc. Natl. Acad. Sci. USA 94, 7857-7861.-   22. Stewart, J. D. and Benkovic, S. J. (1995) Nature 375, 388-391.-   23. Tramontano, A. (1994) Appl. Biochem. Biotechnol. 47, 257-275.-   24. Rao, G. and Philipp, M. (1991) J. Protein Chem. 10, 117-122.    Zhou, G. W., Guo, J., Huang, W., Fletterick, R. J. and    Scanlan, T. S. (1994) Science 265, 1059-1064.    Footnotes for Example I-   ² Abbreviations: Ab, antibody; DFP, diisopropyl fluorophosphate;    EAR-MCA, N-tert-butoxycarbonyl- -benzyl-Glu-Ala-Arg    4-methylcoumaryl-7-amide hydrochloride; ESI, electrospray    ionization; FU, fluorescence unit; L chain, light chain; MALDI-TOF    MS, matrix-assisted laser desorption ionization time-of-flight mass    spectrometry; TA, transition state analog; t_(R), retention time;    VPR-MCA, tert-butoxycarbonyl-Val-Pro-Arg 4-methylcoumaryl-7-amide.-   ³ For example, the apparent first-order rate constant for hydrolysis    of monophenyl methanephosphonate (2.0 mM) in 2.12 M aq. NaOH at    39° C. is 0.228×10⁻³ min⁻¹, corresponding to a half life of 50 h,    whereas hydrolysis of diphenyl esters by equimolar NaOH is complete    within minutes.-   ⁴ Second-order rate constants (M⁻¹ min⁻¹) of p-nitrophenyl    methanephosphonate, which contains a more reactive leaving group    than 2-4, are on the order of 10⁻¹¹-10⁻⁹ for hydrolysis in buffered    solutions (pH 7.6, 30° C.) and 10⁻⁷-10⁻³ in alkaline solutions (pH    8-10, 30-50° C.).

⁵Observed K_(i)/K_(m) are frequently unequal to k_(cat)/k_(uncat).Simple charge complementarity, therefore, does not explain the observedrate accelerations.

EXAMPLE II Phosphonate Ester Probes for Proteolytic Antibodies

Abs and Ab L chains are reported to catalyze the cleavage of VIP₂ (1,2),the HIV coat proteins gp41 (3) and gp120 (4), Arg-vasopressin (5),thyroglobulin (6), factor VIII (7), prothrombin (8) and various modelpeptidase substrates (5,9,10). Recent studies suggest that the peptidaseactivity is a heritable function encoded by a germline V region gene(s)(11,12). In principle, the immune system may be capable of recruitingthe catalyst-encoding germline V gene(s) to elaborate specificproteolytic Abs directed to diverse polypeptide antigens, much likenoncatalytic Abs capable of high affinity binding to different antigenscan be developed by somatic sequence diversification of the samegermline V genes. Introduction of single replacement mutations in Abcombining sites can result in gain of proteolytic (13) and esteraseactivities (14), underscoring the potential contributions of V regiondiversification in maturation of Ab catalytic activities.

The presence of a serine protease-like catalytic triad in a modelproteolytic Ab L chain has previously been deduced from site-directedmutagenesis studies (15). Formation of a covalent complex between thenucleophilic serine residue and the substrate (the acyl-enzymeintermediate) is an essential step en route to peptide bond cleavage bynon-Ab serine proteases (16). Phosphonate diesters, like the classicalinhibitor DFP, can bind the active site of non-Ab serine proteases andserine esterases covalently (17-19).

In comparison, negatively phosphonate monoesters have traditionally beenassumed to bind esterolytic Abs (20,21) and non-Ab serine esterases (22)via noncovalent electrostatic interactions. The aim of the present studywas to characterize the reactivity of recombinant proteolytic Abs withphosphonate diesters and monoesters. Irreversible, active-site directedinhibition of catalytic activity by the phosphonate diesters wasevident, stable Ab-phosphonate ester adducts were resolved underdenaturing conditions; and, the catalytic activity was enriched bychemical selection of phage displayed Ab libraries on immobilizedphosphonate esters. Although less potent, a phosphonate monoesterdisplayed a reactivity profile similar to the diester, suggesting thatAb covalency contributes to the monoester binding. These observationshelp establish the serine protease character of natural Ab catalysts,offer a means towards isolation of efficient catalysts, and offer anexplanation for unexpected catalytic mechanisms encountered in the caseof certain esterase Abs raised by immunization with phosphonatemonoesters.

Experimental Methods

Phosphonate esters: The asymmetric diester I (FIG. 8) was prepared intwo steps: condensation of phenylphosphinic acid and tropine by means oficyclohexylcarbodiimide in dichloromethane and the subsequent oxidationin the presence of p-nitrophenol and triethylamine (18,19). Biotinylateddiester II was prepared from compound I by alkylation withiodoacetyl-LC-biotin (Pierce) in DMF at 60° C. for 4 h. Monoester IIIwas prepared from Sulfo-NHS-SS-biotin (Pierce) and phenylamino(4-amidinophenyl)methanephosphonate. Diester IV was prepared frombiotin disulfide N-hydroxysuccinimide ester (Sigma) and diphenylamino(4-amidinophenyl)methanephosphonate. All products were purified byRP-HPLC and characterized by ₁H-NMR and electrospray ionization massspectrometry. Stock solutions of the compounds were in 30% acetonitrile.

Peptidase assay: Synthetic VIP (HSDAVFTDNYTRLRKQMAVK KYLNSILN-NH2 (SEQID NO: 3); Bachem, Torrance, Calif.) was radiolabeled with 125 I and(Tyr 10-125 I)VIP was separated by RP-HPLC (23). Hydrolysis of (Tyr10-125 I)VIP treated with Abs was determined and corrected forbackground radioactivity in the absence of catalyst (3-15% of availableradioactivity) (24). Preliminary assays showed the cleavage of (Tyr10-125 I)VIP (0.2 nM) to be a linear function of catalyst concentration.Kinetic constants were computed by fitting rate data to theMichaelis-Menten equation v=(Vmax.[S])/(Km+[S]) (25). Cleavage ofpeptide-MCA conjugates (Peptides International) by antibody fragmentswas assayed fluorimetrically in 96-well plates (11). Backgroundfluorescence of substrate in diluent was <10 FU. DFP and other proteaseinhibitors were from Sigma.

Recombinant Abs: Preparation of somatically matured L chain clone c23.5(15; GenBank accession 896288), germline L chain clone c23.5 (12), Lchain clone hk14 (25; GenBank accession 1850134) and the single chain Fvclone mRT3 (26) has been described. Unless otherwise specified, datareported here are for the mature L chain c23.5. Standard methods (25-27)were applied to prepare these libraries: (a) human lupus L chains clonedin pCANTAB5his6 vector (from 3 patients); (b) human lupus single chainFv constructs in pHEN2 (from 2 patients; vector kindly provided byCentre for Protein Engineering, MRC, England; patent WO9201047-A,GenBank accession 1926701); (c) L chains from a mouse immunized with VIPin pCANTAB5his6 [source details in ref. 28; sequence of 2 L chains fromthis library available in GenBank in (U4 and U16, accession AF329094 andAF329095, respectively]; and (d) single chain Fv constructs from miceimmunized with the extracellular domain of epidermal growth factorreceptor in pCANTAB5his6 (immunization method to be describedelsewhere). The murine and human Fv libraries were cloned asVH-linker-VL [linker: (GGGGS)3] and VL-linker-VH [linker:SS(GGGGS)2GGSA)] constructs, respectively. Both Fv orientations supportantigen-binding (25,29)]. Following hypotonic lysis of erythrocytes inmurine splenocytes (2×10⁷ cells) or peripheral blood leukocytes (from100 ml blood), total RNA was isolated, a cDNA copy prepared usingforward primers, and the cDNA for full-length L chains and the VH, Vκ,and Vλ domains was prepared by PCR (corresponding to residues 1-214;1-123; 1-107; and, 1-107, respectively; Kabat numbering). Mouse Fvprimers are described by Pharmacia (Recombinant Antibody Manual).

Primers for remaining libraries are:

-   (a) Human full-length L chain: VLK back (Sfi 1 site underlined)—-   GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGACATCCAGATGACCCAGTCTCC (SEQ ID NO:    4),-   GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGATGTTGTGATGACTCAGTCTCC (SEQ ID NO:    5),-   GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGAAATTGTGTTGACGCAGTCTCC (SEQ ID NO:    6),-   GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGACATCGTGATGACCCAGTCTCC (SEQ ID NO:    7),-   GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGAAACGACACTCACGCAGTCTCC (SEQ ID NO:    8),-   GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGAAATTGTGCTGACTCAGTCTCC (SEQ ID NO:    9),-   Ck forward (Not I site underlined)—-   CCATCCTGCGGCCGCACACTCTCCCCTGTTGAAGCTCTT (SEQ ID NO: 10);-   (b) Human single chain Fv: VLk back B see back primers, full-length    L chain; VLk forward (Xho I site underlined) B-   GCCTGAACCGCCTCCACCACTCGAGCGTTTGATTTCCACCTTGGTCCC (SEQ ID NO:11),-   GCCTGAACCGCCTCCACCACTCGAGCGTTTGATCTCCAGCTTGGTCCC(SEQ ID NO: 12),-   GCCTGAACCGCCTCCACCACTCGAGCGTTTGATATCCACTTTGGTCCC (SEQ ID NO: 13),-   GCCTGAACCGCCTCCACCACTCGAGCGTTTGATCTCCACCTTGGTCCC (SEQ ID NO: 14),-   GCCTGAACCGCCTCCACCACTCGAGCGTTTAATCTCCAGTCGTGTCCC (SEQ ID NO: 15);-   VLk back (Sfi I site underlined) B-   GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGTCTGTGTTGACGCAGCCGCC (SEQ ID NO:    16),-   GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGTCTGCCCTGACTCAGCCTGC (SEQ ID NO:    17),-   GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCTCCTATGTGCTGACTCAGCCACC (SEQ ID NO:    18),-   GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCTCTTCTGAGCTGACTCAGGACCC (SEQ ID NO:    19),-   GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCACGTTATACTGACTCAACCGCC (SEQ ID NO:    20),-   GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGCTGTGCTCACTCAGCCGTC (SEQ ID NO:    21),-   GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCAATTTTATGCTGACTCAGCCCCA (SEQ ID NO:    22);-   VLk forward (Xho I underlined) B-   GCCTGAACCGCCTCCACCACTCGAGCCTAGGACGGTGACCTTGGTCCC (SEQ ID NO: 23),-   GCCTGAACCGCCTCCACCACTCGAGCCTAGGACGGTCAGCTTGGT CCC (SEQ ID NO: 24),-   GCCTGAACCGCCTCCACCACTCGAGCCTAAAACGGTGAGCTGGGTCCC (SEQ ID NO: 25);-   CL_forward B-   TGAAGATTCTGTAGGGGCCACTGTCTT (SEQ ID NO: 26);-   VH back (ApaL site underlined) B-   CATGACCACAGTGCACTTCAGGTGCAGCTGGTGCAGTCTGG (SEQ ID NO: 27),-   CATGACCACAGTGCACTTCAGGTCAACTTAAGGGAGTCTGG (SEQ ID NO: 28),-   CATGACCACAGTGCACTTGAGGTGCAGCTGGTGGAGTCTGG (SEQ ID NO: 29),-   CATGACCACAGTGCACTTCAGGTGCAGCTGCAGGAGTCGGG (SEQ ID NO: 30),-   CATGACCACAGTGCACTTCAGGTGCAGCTGTTGCAGTCTGC (SEQ ID NO: 31),-   CATGACCACAGTGCACTTCAGGTACAGCTGCAGCAGTCAGG (SEQ NO: 32);-   VH forward (Not I site underlined) B-   GAGTCATTCTGCGGCCGCGGGGAAGATGGGCCCTTGGT (SEQ ID NO: 33),-   GAGTCATTCTGCGGCCGCGGGGAAAAGGGTTGGGGCGGATGC (SEQ ID NO: 34);-   (c) Mouse full-length L chain: VLk back (Sfi I site underlined) B-   GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGATGTTTTGATGACCCAAACTCCA (SEQ ID    NO: 35),-   GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGATATTGTGATAACCCAGGATGAA (SEQ ID    NO: 36),-   GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGACATTGTGCTRACCCAGTCTCCA (SEQ ID    NO: 37),-   GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGACATCCAGATGACNCAGTCTCCA (SEQ ID    NO: 38),-   GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAAATTGTTCTCACCCAGTCTCCA (SEQ ID    NO: 39),-   GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGAAAATGTGCTCACCCAGTCTCCA (SEQ ID    NO: 40);-   C_forward (Not I site underlined) B-   GAGTCATTCTGCGGCCGCCTCATTCCTGTTGAAGCTCTTGAC (SEQ ID NO: 41)    Linkage of mouse VL/VH domains was according to Pharmacia. Cloning    of human Fv library in pHEN2 was by a two-step procedure—VH cDNA    insertion via the ApaLI/NotI sites, and VL cDNA insertion via the    SfiI/XhoI sites. Library sizes were—murine L chain, 1.2×10⁵; murine    Fv, 3.3×10⁷; human L chain, 1.2×10⁶; human Fv, 1.4×10⁷. Randomly    picked clones (at least five from each library) were sequenced by    the dideoxy nucleotide sequencing method; 100, 75, 100 and 60% of    the clones, respectively, contained full-length, stop codon-free,    non-identical sequences.    Phage selection and Ab purification: Phage particles displaying Ab    fragments as g3 fusion proteins (2-5×10¹³ CFU) treated with    biotinlyated phosphonate esters (100 μl binding buffer, 50 mM    sodiumphosphate, pH 8.0; 30 min, 37° C.) were precipitated with    PEG (30) and adsorbed (60 min) on streptavidin coated Immunotubes (2    μg/tube, blocked with 5% BSA). The tubes were washed 4× with binding    buffer containing 0.05% Tween-20 and 0.5 M NaCl; 4× with 0.1 M    glycine-HCl, pH 2.7, 0.05% Tween-20; and 2× with binding buffer    containing 0.05% Tween-20. Bound phages were eluted with 20 mM 2-PAM    (24 hours, 25° C.) or 10 mM 2-mercaptoethanol (30 min, 25° C.) in 50    mM sodium phosphate, pH 8.0, respectively. Soluble Ab fragments in    the periplasmic extracts of HB2151 cells were quantified by    dot-blotting for the c-myc tag (30; expression level 0.4-6    mg/liter). Purification was by IMAC (31). For initial screening, the    extracts (0.6 ml) were dialyzed against the column binding buffer    and subjected to one round of IMAC (Bio-Spin columns, BioRad; 50 μl    Ni-NTA agarose gel, Qiagen; elution with 0.25 ml pH 5 buffer). Large    scale purifications from 250 ml cultures were carried out by two    IMAC rounds.

SDS-gel electrophoresis (Phast gels 8-25%) showed a major 27 kD silverstained recombinant protein that was immunoblottable with anti-c-mycantibody (corresponding to Fv and L chain monomers). Some preparationscontained a minor 55 kD dimer band and a 17 kD C-terminal fragment ofthe recombinant proteins, both of which were stained with anti-c-mycantibody (the fragment contains the C-terminal metal binding poly(his)tag and copurifies with the full-length proteins, ref 15).Non-denaturing gel filtration (2) of purified Ab fragments yielded theproteins as a major 27-28 kD peak with peptidase activity essentiallyidentical to the preparations loaded on the column. In the case of cloneYZ17 Fv, higher order aggregation was evident in preparations purifiedby IMAC. Further purification was by anion exchange chromatography on aMono-Q column (Pharmacia; 0-0.5 M NaCl, 30 min), yielding anelectrophoretically homogeneous 27-28 kD Fv fraction eluting at 0.37MNaCl.

Phosphonate ester-Ab binding: Complex formation was monitored by gelfiltration (Superose-12 column, Pharmacia; 0.5 ml/min in 50 mM Tris-HCl,100 mM glycine, pH 8, 0.15 M NaCl, 0.025% Tween-20, 0.02% sodium azide).The column was calibrated with thyroglobulin, IgG, albumin, ovalbuminand ribonuclease (Pharmacia). ELISA for bound phosphonate ester wasincubation of column fractions in streptavidin coated 96-well plates (60min, 37° C., React-Bind™; blocked with Blocker™ SA, Pierce). Unboundprotein was removed by washing with 0.1 M sodium phosphate, pH 7.4, 0.1%BSA, 0.025% Tween-20 (PBS-T), and adducts were quantified using mouseanti-c-myc (60 min; 100 μl, 1:500 ascites fluid from hybridoma 9EI0,ATCC) and peroxidase conjugated goat anti-mouse IgG (_(Fc) specific,1:1000; Sigma) (30). Phosphonate ester binding data reported here arefor the monomer protein fraction recovered by gel filtration. Thebinding was also determined by a dot blot method after separatingadducts and free phosphonate esters by gel filtration (Biospin 6columns, BioRad, exclusion limit 6 kD). The excluded fraction (50 μl)was allowed to pass through a nitrocellulose membrane (0.2 μm;Trans-Blot; Biorad) using a 96-well blotting apparatus (Biorad), themembrane blocked with 5% nonfat milk, treated with peroxidase conjugatedstreptavidin (1:1000, Sigma) in PBS-T, and bound phosphonate estersdetermined using an electrochemiluminscence kit (Amersharn-Pharmacia)and X-OMAT film (Kodak). For SDS-polyacrylamide gel electrophoresis(4-20% gels), the Ab fragments were incubated with phosphonate esters(30 min), the complexes recovered by precipitation with 10% TCA, thepellets redissolved in electrophoresis buffer containing 2% SDS and 5 mM2-mercaptoethanol, boiled for 2 min and subjected to electrophoresis.The gels were electroblotted onto nitrocellulose membranes (Transblot;Biorad), the blots treated overnight with 5% nonfat milk in PBS-T,washed with PBS-T, and biotinylated bands detected as described for thedot blots.

Results

Phosphonate diester reactivity of Abs. VIP cleavage by anti-VIP L chainclones hk14 and c23.5 (germline and somatically matured proteins) hasbeen described (12,15,27). In initial studies, additional proteolytic Abfragments were identified to study the inhibitory effect of DFP andphosphonate diester II (FIG. 8). Nineteen randomly picked L chain clones(from a mouse immunized with VIP, U series clones) and an Fv cloneisolated by McAfferty and coworkers by binding of phage Abs to aphosphonate monoester (mRT3, ref 26) were screened for VIP cleavingactivity. Seven L chains and the Fv displayed (Tyr₁₀-₁₂₅I)VIP cleavingactivity (>10% cleavage; catalyst concentration 20 nM). Confirmation ofthe peptidase activity as belonging to the Ab fragments was based on theobserved electrophoretic homogeneity of the catalysts, elution of theactivity in the 27-28 kD light chain monomers purified by gel filtration(2), and the absence of detectable peptidase activity in equivalentlypurified extracts from several L chain clones (e.g., clone U21) andextracts from bacteria containing the control vector (no Ab insert). DFPand diester II at concentrations of 1 mM and 0.1 mM, respectively,inhibited the catalytic activity of every Ab fragment analyzed by atleast 40% (n=11 and 6 clones, respectively). See FIG. 9. Near-equivalentinhibition of the activities of the germline and mature forms of L chainc23.5 by II was observed. The inhibitory effect was relieved to varyingextent at 5-fold lower DFP and II concentrations in every case.Inhibition was also evident using a peptide-MCA substrate [83, 71, 92and 79% inhibition of cleavage of PFR-MCA (200 μM) by II (50 μM) usingas catalysts L chain c23.5, L chain hk14, Fv mRT3 and L chain U16;catalyst concentration 0.5 μM]. Removal of free II from the Ab reactionmixture by gel filtration did not restore the catalytic activity,suggesting that the inhibition was irreversible (FIG. 10). The substrate(VIP) protected against II inhibition of catalytic activity. II containsa biotin tag, permitting determination of its its binding to the Abfragments with a streptavidin-peroxidase conjugate. II-labeled Fv wasresolved by gel filtration (Superose-12 column) as a biotin-containing27-28 kD peak coeluting with the A280 optical density peaks of Fvmonomers (FIG. 11A). Inclusion of excess I in the reaction mixture (10mM; I is the nonbiotinylated form of II) inhibited the labeling by >90%.Essentially similar results were observed by gel filtration ofII-containing adducts of L chain clones c23.5, hk14 and U16 (labeling ofthe monomer L chains was at 18, 33 and 51% of the Fv level in FIG. 11,respectively). SDS-electrophoresis of reaction mixtures followed bystaining with streptavidin-peroxidase permitted identification of II-Abfragment adducts at the correct mass corresponding to the Fv and L chainmonomers (FIG. 11B). II binding by the Fv was reduced in the presence ofthe substrate (FIG. 11C). Control extracts of bacteria harboring vectorwithout an Ab insert did not bind II. These observations indicateirreversible, active site-directed II binding by the Ab fragments.

Covalent phage selection. A compound similar to diester II is describedto permit isolation of catalytically active subtilisin mutants from aphage library (32). Monoester III-like compounds are generally thoughtbind esterolytic Abs by noncovalent electrostatic interactions (20,21).However, Fv mRT3, which had been initially identified based on bindingto a phosphonate monoester (26), also displayed the ability to binddiester II irreversibly (FIG. 11). Therefore, both diester II andmonoester III were analyzed for the ability to capture phage displayedcatalytic Abs. II- and III-phage Ab complexes are trapped on immobilizedstreptavidin via the biotin tag. Noncovalently bound phages were removedby exhaustive washing at pH 2.7 (and additional pH 12 washes with 0.1 Mtriethylamine in the case of selection with monoester III). Elution ofII-bound phages was by treatment with 2-PAM, which is described tocleave the P-O bind in DFP-serine esterase adducts (33), and ofIII-bound phages, by reduction of the S—S bond in III. The Ab fragmentsencoded by selected phagemid DNA were expressed in soluble form,purified by IMAC and analyzed for catalytic activity along with thecontrol unselected Abs. To determine selection efficacy, cleavage of themodel substrate PFR-MCA was measured at equivalent concentrations ofpolyclonal selected and unselected Ab fragments (FIG. 12). About 10-40fold enrichment of the PFR-MCA cleaving activity was evident in theselected Abs from the human lupus L chain, human lupus Fv and murine Fvlibraries. Increasing levels of catalytic activity were evident as afunction of increasing length of elution of II-bound phages with PAM (20and 70 FU/16 h/μM lupus light chains following elution for 3 and 24hours, respectively) and increasing concentration of 2-mercaptoethanolapplied for elution of III-bound phages (FIG. 12B). Essentially similarlevels of catalyst enrichment were evident in the selected Abpopulations using VIP as substrate (not shown). II-selected L chainsdisplayed increased II binding (by 11-fold) compared to unselected Lchains, analyzed by gel filtration and detection of adducts by ELISA asin FIG. 9. Taken together, these studies demonstrate the ability ofdiester 11 and monoester III to preferentially bind the catalyst subsetpresent in the phage Abs.

Individual catalytic Fv and L chains from the selected populations wereidentified by screening for peptidase activity. Sixty percent ofIII-selected Fv clones and 69% of II-selected L chain clones displayedPFR-MCA cleaving activity >2 FU/h/μM above the background level(activity of equivalently purified periplasmic extracts from bacteriaharboring vector without an Ab insert; substrate 200 μM). Preferentialcleavage of peptide-MCA substrates on the C terminal side of a basicresidue (Arg) by a II-selected light chain (clone GG63) and aIII-selected Fv (clone YZ17) was evident (FIG. 13). Cleavage ofsubstrates containing MCA linked to acidic or neutral residues wasundetectable. Saturation kinetics consistent with theMichaelis-Menten-Henri equation were observed for both clones usingtheir preferred peptide-MCA substrates (YZ17 Fv, EAR-MCA; GG63 L chain,PFR-MCA; Table 1). DFP inhibited the proteolytic activity of both Abfragments, confirming the mechanistic class of the catalysts (Table 1).Inhibitors of other protease classes did not influence the reactiondetectably (iodoacetamide, phenanthroline, pepstatin A). Interestingly,diester IV inhibited the catalytic activity of Fv YZ17 more potentlythan monoester III Fv YZ17 Fv, even though this clone had been isolatedby binding the monoester (FIG. 14). Moreover, Fv YZ17 was stainedstrongly by diester IV assessed by SDS-electrophoresis, whereas stainingwith monoester III was barely visible (FIG. 15). L chain GG63, which hadbeen isolated by binding to diester II was also stained more strongly bydiester IV compared monoester III. SDS-electrophoresis of a crudeperiplasmic extract of Fv clone YZ17 treated with diester II revealed asingle biotinylated band corresponding to 27-28 kD Fv. Other periplasmicproteins in the extract were not stained, and staining of periplasmicproteins in a control extract of bacteria harboring vector without anantibody insert was not evident. It may be concluded that phosphonatediesters and monoesters bind the proteolytic Abs irreversibly, with themonoester displaying a lower level of reactivity.

According to their cDNA sequences, light chain clone belongs to family1, subgroup I (Kabat database). The VL and VH domains of Fv cone YZ17(GenBank AF329093, Appendix 2) belong to family XXVI, subgroup V andsubgroup I, respectively (VH family designated ‘Miscellaneous’ in Kabatdatabase). The VL domains of GG63 and YZ17 contain 17 and 2 amino acidsubstitutions compared to their germline gene counterparts, respectively(GenBank accession numbers 33197 and 5305062, respectively). Thegermline counterpart of YZ17 VH domain could not be determined withcertainty.

TABLE I Inhibition, % K_(m), Phenan- Iodo- Catalyst μM k_(cat), min⁻¹DFP throline acetamide Pepstatin A Fv YZ17 10.1 0.5 (0.09) 84.6 0.2 4.41.2 (2.9) (1.2) (3.3) (2.3) (3.1) L chain 7.9 0.2 (0.05) 74.4 0.6 5.80.2 (1.3) GG63 (2.4) (0.1) (0.7) (4.5)Table 1: Apparent kinetic constants and inhibition profile ofCRAA-selected antibody fragments. Substrate, EAR-MCA (YZ17 Fv) andPFR-MCA (GG63 light chain). Values in parentheses, standard deviation.Rate data at increasing substrate concentration (2.5, 5, 10, 20, 40, 80,160 Mm) were gathered over 7 hours and fitted to the Michaelis-Mentenequation. Reaction conditions 7 hours, 37° C. Inhibition was assayed bypreincubation of catalysts with various protease inhibitor # (30 min,37° C.), addition of substrate to 200 μM, and measurement of reactionrate over 5 hours. DFP 0.5 mM; phenanthroline 10 mM; iodacetamide 0.5mM; pepstatin A 10 μM. Fv YZ17 40 nM. L chain GG63 80 nM. Inhibitiondata are expressed as percent of fluorescence in the absence ofinhibitor (Fv YZ17, 93 FU; L chain GG63, 71 FU).Discussion

Formation of the tetrahedral transition state of peptide bond cleavageby non-Ab serine proteases involves covalent attack by the active sitenucleophile and transfer of a negative charge to the carbonyl oxygen inthe substrate. The phosphonate diester group mimics the substratecarbonyl, and the phosphorous atom is sufficiently electrophilic topermit covalent binding to the active site of serine proteases (18,19).Phosphonate diesters have recently been applied to isolate mutants of anon-Ab serine protease (subtilisin) displayed on a a phage displaylibrary (32). A positive charge was placed close to the phosphonateesters utilized as probes for proteolytic Abs in the present study,because most known proteolytic antibodies cleave peptide bonds on theC-terminal side of basic residues (3,5-8,31). In FIG. 9, identificationof Ab fragments as proteases had been achieved without reliance on themechanistic class of the catalysts (i.e., by binding to VIP, randomscreening for catalytic activity). Yet, the diester inhibited theproteolytic activity of all six Abs analyzed. The inhibition wasirreversible and active site-directed. Formation of stable adducts ofthe phosphonate diester with proteolytic L chains and a Fv construct wasevident by column chromatography and denaturing electrophoresis.Evidently, therefore, a serine protease-like mechanism is recurrentlyencountered in Abs expressing proteolytic activity. The phage selectionsconfirmed this conclusion. As predicted, selection of the phages bybinding to the diester enriched the proteolytic activity.

Results from phosphonate monoester studies presented in Example I offerinteresting insights to certain aspects of Ab catalysis. McAfferty etal. (26) isolated Fv mRT3 by immunization and phage selection with aphosphonate monoester. This Fv was observed in the present study to binddiester II irreversibly and to express proteolytic activity. Phosphonatemonoesters have previously been advanced as oxyanionic transition stateanalogs that bind catalytic Ab active sites solely by noncovalent means.[The electrophilicity of the monoesters is generally held to be too weakto permit covalent binding to the active site]. On the other hand,catalytic mechanisms that are not predicted from models of simplenoncovalent binding interactions have been encountered in Abs followingimmunization with phosphonate monoesters (reviewed in ref 34), forinstance an anti-monoester Ab with an apparently fortuitous serineesterase activity (35). In the present study, the utility of phosphonatemonoesters in selecting proteolytic L chains and Fv has been confirmeddirectly, as is evident from the enriched catalytic activity ofmonoester IlI-selected Ab populations in FIG. 12. Remarkably, Fv cloneYZ17 selected by binding to monoester III was even more reactive withthe diester analog IV. Monoester III also formed stable adducts with aclassical serine protease, trypsin, and it inhibited enzyme activityirreversibly. It may be concluded that the monoester can mimic thetransition state of peptide bond hydrolysis by virtue of its covalentreactivity in addition to the oxyanionic character. These observationsare not without precedent. A survey of the literature has identified tworelevant reports. One describes the covalent reactivity of phosphonatemonoesters with P-lactamase based on inspection of inhibition kinetics(36; this enzyme utilizes an active site serine nucleophile forcatalysis). The second reports covalent binding of a phosphonic acidcompound to a cholineesterase (37; phosphonic acid has two negativecharges—its reactivity with active site nucleophiles is predicted to beeven lower than the monoester). It appears safe to conclude, therefore,that covalent binding to the active site may, in part, underlie theability of the phosphonate monoester to select proteolytic Abs from thelibraries. The foregoing conclusions are not in conflict with an earlyreport describing the failure to isolate Phe-Ile or Ile-Gly cleavingmonoclonal IgG Abs from mice immunized with a phosphonate monoester(38). The catalysts identified in the present report display specificityfor cleavage on the C terminal side of basic residues, which may accountfor lack of cleavage at peptide bonds linking neutral residues.Moreover, the methods applied in ref 38 were designed to identify Abscapable of noncovalent phosphonate monoester binding, whereas our phageselection protocols were biased to enrich covalently reactive Abs at theexpense of noncovalent binders. There is also the issue of structuraldifferences remote from the active site—ref 38 studied full-length IgGwhereas recombinant single chain Fv and L chain subunits were analyzedin the present study. The catalytic characteristics of IgG, Fv and the Lchain subunit can differ profoundly (28,31,39). Ab combining sites canbe quite diverse because of the existence of multiple germline V genesand somatic mechanisms permitting sequence diversification of the Vdomains (V-J/V-D-J rearrangement; CDR hypermutability). Whether a singlephosphonate ester structure can serve as an efficient binding reagentfor proteolytic Abs with different substrate specificities depends onthe extent of active site conservation. Synthetic peptides correspondingto antigen regions distant from the cleavage site can inhibitproteolytic Abs (40). Moreover, mutations that decrease binding to theantigen ground state do not decrease the rate of catalysis by an Ab Lchain (15), suggesting the existence of distinct subsites responsiblefor the chemical reactivity and initial antigen binding (correspondingto the transition state and ground state stabilization steps,respectively). A comparatively conserved catalytic subsite may becompatible, therefore, with differing Ab specificities for individualantigens derived from distinct noncovalent interactions at the groundstate binding subsite. This model is consistent with preservation of agermline-encoded catalytic subsite in the V domain over the course of Bcell clonal selection, even as antigen binding affinity improves due toremote mutations. Note, however, that the chemical reactivity of activesite nucleophiles is determined in part by intramolecular interactions,for instance, by formation of hydrogen bonds between Ser, His and Aspresidues (16). Depending on the specific structural changes introducedby V domain sequence diversification, improvements or deteriorations ofthe catalytic machinery are feasible. The level of catalytic activityexpressed by Abs may depend in part, therefore, upon the immunologicalhistory of the repertoires employed as the source of the phagelibraries. In particular, the somatically mature repertoire in certainautoimmune and lymphoproliferative disorders frequently expresses Abcatalysts (6,8,41-43), and may be suitable for isolating Ab catalysts.The phage experiments in the present study were intended to study thephosphonate ester reactivity of Abs, as opposed to isolating the highestactivity catalysts. Whether chemical isolation of medically usefulcatalysts is feasible will depend on further technological improvments.The turnover numbers for phosphonate ester-selected Fv YZ17 and L chainGG63 are greater than observed for proteolytic Ab fragments identifiedby random screening (9,10). More refined phage selection protocols mayhelp identify high turnover, antigen-specific proteolytic Abs, e.g.,repeated rounds of selection using phosphonate esters flanked byappropriate peptide sequences. The phosphonate esters could also beapplied as immunogens to elicit the synthesis of serine protease-likeAbs on demand, assuming that covalent antigen binding by Abs on thesurface of B cells can drive clonal selection and Ab affinitymaturation. A similar strategy has been previously been proposed toelicit the synthesis of aldolase Abs (44) The pitfall, of course, isthat irreversible immunogen binding by B cells may tolerize the cells,as is believed to occur upon persistent occupancy of the B cell receptorby noncovalent immunogens (45).

GenBank Accession numbers and information for U4L chain, U16L chain,YZ17Fv and G63 L chain are provided below.

GenBank Accession No. and Information for U4L chain

AF329094. Mus musculus dome...[gi:12751402]LOCUS    AF329094         330 bp    mRNA   linear  ROD Jul. 24, 2001DEFINITION Mus musculus domesticus U4 recombinant antibody light chainVL      domain mRNA, partial cds. ACCESSION  AF329094VERSION   AF329094.1 GI:12751402 KEYWORDS SOURCE  western European housemouse.  ORGANISM Mus musculus domesticus      Eukaryota; Metazoa;Chordata; Craniata; Vertebrata; Euteleostomi;      Mammalia; Eutheria;Rodentia; Sciurognathi; Muridae; Murinae; Mus. REFERENCE 1 (bases 1 to330)  AUTHORS Paul,S., Tramontano,A., Gololobov,G., Zhou, Y.X.,Taguchi,H.,      Karle,S., Nishiyama, Y., Planque,S. and George,S. TITLE  Phosphonate ester probes for proteolytic antibodies  JOURNAL J.Biol. Chem. 276 (30), 28314-28320 (2001)  MEDLINE 21359358 PUBMED  11346653 REFERENCE 2 (bases 1 to 330)  AUTHORS Paul,S., Zhou,Y.-X., Nishiyama, Y., Taguchi,H., Karle,S.,      Gololobov,G.,Planque,S., George,S. and Tramontano,A.  TITLE  Direct Submission JOURNAL Submitted (Dec. 14, 2000) Pathology and Laboratory Medicine,     University of Texas-Houston Medical School, 6431 Fannin, Houston,     TX 77030, USA FEATURES        Location/Qualifiers  source    1..330           /organism=“Mus musculus domesticus”          /strain=“BALB/c”           /sub_species=“domesticus”          /db_xref=“taxon:10092”           /clone=“U4”          /cell_type=“B-lymphocyte”          /tissue_type=“hyperimmunized spleen”          /dev_stage=“young adult”           /note=“mice immunized withVIP-KLH conjugate”   CDS       <1..>330           /codon_start=1          /product=“recombinant antibody light chain VL domain”          /protein_id=“AAK07643.1”           /db_xref=“GI:12751403”/translation=“DVLMTQSPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FQGSHVPYTFGGGQTGK(SEQ ID NO: 42)” BASE COUNT  87 a  79 c  79 g  85 t ORIGIN   1gatgttttga tgacccaatc tccactctcc ctgcctgtca gtcttggaga tcaagcctcc  61atctcttgca gatctagtca gagcattgta catagtaatg gaaacaccta tttagaatgg 121tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 181tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 241agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttccg 301tacacgttcg gagggggcca aactggaaaa (SEQ ID NO: 43) GenBank Accessionnumber and information for U16 L chain AF329095. Mus musculusdome...[gi:12751404] LOCUS    AF329095          307bp   mRNA   linear  ROD Jul. 24, 2001 DEFINITION Mus musculus domesticusU16 recombinant antibody light chain VL      domain mRNA, partial cds.ACCESSION  AF329095 VERSION   AF329095.1 GI:12751404 KEYWORDSSOURCE   western European house mouse.  ORGANISM Mus musculus domesticus     Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;     Mammalia; Eutheria; Rodentia; Sciurognathi; Muridae; Murinae; Mus.REFERENCE 1 (bases 1 to 307)  AUTHORS Paul,S., Tramontano,A.,Gololobov,G., Zhou, Y.X., Taguchi,H.,     Karle,S., Nishiyama, Y.,Planque,S. and George,S.  TITLE Phosphonate ester probes for proteolyticantibodies  JOURNAL J. Biol. Chem. 276 (30), 28314-28320 (2001)  MEDLINE21359358  PUBMED  11346653 REFERENCE 2 (bases 1 to 307)  AUTHORSPaul,S., Zhou, Y.-X., Nishiyama, Y., Taguchi,H., Karle,S.,     Gololobov,G., Planque,S., George,S. and Tramontano,A. TITLE  Direct Submission  JOURNAL  Submitted (Dec. 14, 2000) Pathologyand Laboratory Medicine,      University of Texas-Houston MedicalSchool, 6431 Fannin, Houston,      TX 77030, USAFEATURES        Location/Qualifiers   source   1..307          /organism=“Mus musculus domesticus“           /strain=“BALB/c”          /sub_species=“domesticus”           /db_xref=“taxon:10092”          /clone=“U16”           /cell_type=“B-lymphocyte”          /tissue_type=“hyperimmunized spleen”          /dev_stage=“young adult”           /note=“mice immunized withVIP-KLH conjugate”   CDS       <1..>307           /codon_start=1          /product=“recombinant antibody light chain VL domain”          /protein_id=“AAK07644.1”           /db_xref=“GI:12751405”/tranlaslation=“QNVLTQSPALMSASPGEKVTITCSASPSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSY PWTFGGAPS (SEQ IDNO: 44)” BASE COUNT   74 a  90 c  72 g  71 t ORIGIN   1 caaaatgttctcacccagtc tccagcactc atgtctgcat ctccagggga gaaggtcacc  61 ataacctgcagtgccagccc aagtgtaagt tacatgcact ggttccagca gaagccaggc 121 acttctcccaaactctggat ttatagcaca tccaacctgg cttctggagt ccctgctcgc 181 ttcagtggcagtggatctgg gacctcttac tctctcacaa tcagccgaat ggaggctgaa 241 gatgctgccacttattactg ccagcaaagg agtagttacc cgtggacgtt cggtggagca 301 ccaagct (SEQID NO: 45) GenBank Accession Number and Information for YZ17 FvAF329093. Synthetic constru...[gi:12957377]LOCUS    AF329093            795 bp   mRNA      linear  SYN Jul. 24,2001 DEFINITION Synthetic construct recombinant single-chain Fv antibodymRNA,      partial cds. ACCESSION  AF329093 VERSION   AF329093.1GI:12957377 KEYWORDS SOURCE  synthetic construct.  ORGANISM syntheticconstruct     artifical sequence. REFERENCE 1 (bases 1 to 795)  AUTHORSPaul,S., Tramontano,A., Gololobov,G., Zhou, Y.X., Taguchi,H.,    Karle,S., Nishiyama, Y., Planque,S. and George,S.  TITLE Phosphonateester probes for proteolytic antibodies  JOURNAL J. Biol. Chem. 276(30), 28314-28320 (2001)  MEDLINE 21359358  PUBMED  11346653 REFERENCE 2(bases 1 to 795)  AUTHORS Paul,S., Zhou, Y.-X., Nishiyama, Y.,Taguchi,H., Karle,S.,     Gololobov,G., Planque,S., George,S. andTramontano,A.  TITLE Direct Submission  JOURNAL Submitted (Dec. 13,2000) Pathology and Laboratory Medicine,      University ofTexas-Houston Medical School, 6431 Fannin, Houston,      TX 77030, USAFEATURES        Location/Qualifiers   source   1..795          /organism=“synthetic construct”          /db_xref=“taxon:32630”           /focus   CDS      <1..795          /codon_start=1           /transl_table=11          /product=“recombinant single-chain Fv antibody”          /protein_id=“AAK09206.1”           /db_xref=“GI:12957378”/translation=“QVKLQQSGPELVKPGASVKISCKASGYTFTDYTMDWVKQSHGKSLEWIGYIYPNNGGTGYNQKFKSKATLTVDKSSSTAYMELHSLTSEDSAVYYCARFSSFDYWGQGTTVTVSSGGGGSGGVGSGGGGSDIQMTQSPSSLSASLGDTITITCHASQNINVWLSWYQQKPGNIPKLLIYRASNLHTGVPSRFSGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPLTFGTGTKLEIKRAAAHHHHHHGAAEQKLISEEDLNGAA (SEQ ID NO: 46)”   misc_feature 1..339          /note=“VH domain; Region: antibody heavy chain variable          domain”   source      1..714           /organism=“Mus musculusdomesticus“           /strain=“MRL/Mp-lpr”          /sub_species=“domesticus”           /db_xref=“taxon:10092”          /clone=“YZ-17”           /cell_type=“B-lymphocyte”          /tissue_type=“hyperimmunized spleen”          /dev_stage=“young adult”           /note=“selection with Bt-Zmonoester from mouse library           immunized with exEGFR”  misc_feature  340..390           /note=“Region: linker between VH andVL domains”   misc_feature  391..714           /note=“VL domain; Region:antibody light chain variable           domain”   misc_feature  724..741          /note=“Region: poly-histidine tag”   misc_feature  751..783          /note=“Region: c-myc tag” BASE COUNT      213 a  205 c  199g  178 t ORIGIN   1 caggtgaaac tgcagcagtc aggacctgaa ctggtgaagcctggggcttc agtgaagata  61 tcctgcaagg cttctggtta cacattcact gactacaccatggactgggt gaagcagagc 121 catggaaaga gccttgagtg gattggatat atttatcctaacaatggtgg tactggctac 181 aaccagaagt tcaagagcaa ggccacattg actgtagacaagtcctccag cacagcctac 241 atggagctcc acagcctgac atctgaggac tctgcagtctattactgtgc aagattttcc 301 tcttttgact actggggcca agggaccacg gtcaccgtctcctcaggtgg aggcggttca 361 ggcggagttg gctctggcgg tggcggatcg gacatccagatgactcagtc tccatccagt 421 ctgtctgcat cccttggaga cacaattacc atcacttgccatgccagtca gaacattaat 481 gtttggttaa gctggtacca gcagaaacca ggaaatattcctaaactatt gatctatagg 541 gcttccaact tgcacacagg cgtcccatca aggtttagtggcagtggatc tggaacaggt 601 ttcacattaa ccatcagcag cctgcagcct gaagacattgccacttacta ctgtcaacag 661 ggtcaaagtt atcctctcac gttcggcacg ggcaccaagctggaaatcaa acgggcggcc 721 gcacatcatc atcaccatca cggggccgca gaacaaaaactcatctcaga agaggatctg 781 aatggggccg catag (SEQ ID NO: 47) GenBankAccession Number and Information for G63 L chain AF352557. Homo  sapiensGG-6...[gi:13549147] LOCUS    AF352557            339bp   mRNA      linear  PRI Jul. 24, 2001 DEFINITION Homo Sapiens GG-63immunoglobulin light chain variable region mRNA,      partial cds.ACCESSION  AF352557 VERSION   AF352557.1 GI:13549147 KEYWORDSSOURCE  human  ORGANISM Homo sapiens      Eukaryota; Metazoa; Chordata;Craniata; Vertebrata; Euteleostomi;      Mammalia; Eutheria; Primates;Catarrhini; Hominidae; Homo. REFERENCE 1 (bases 1 to 339)  AUTHORSPaul,S., Tramontano,A., Gololobov,G., Zhou, Y.X., Taguchi,H.,    Karle,S., Nishiyama, Y., Planque,S. and George,S.  TITLE Phosphonateester probes for proteolytic antibodies  JOURNAL J. Biol. Chem. 276(30), 28314-28320 (2001)  MEDLINE 21359358  PUBMED  11346653 REFERENCE 2(bases 1 to 339)  AUTHORS Paul,S., Zhou, Y.-X., Nishiyama, Y.,Taguchi,H., Karle,S.,     Gololobov,G., Planque,S., George,S. andTramontano,A.  TITLE Direct Submission  JOURNAL Submitted (Feb. 22,2001) Pathology and Laboratory Medicine,      University ofTexas-Houston Medical School, 6431 Fannin, Houston,      TX 77030, USAFEATURES        Location/Qualifiers   source   1..339          /organism=“Homo  sapiens”           /db_xref=“taxon:9606”          /clone=“GG-63”           /cell_type=“peripheral bloodlymphocyte”           /dev_stage=“adult”   CDS        <1..>339          /note=“selected on Bt-X (phenylphosphonate 4-nitrophenyl          [N-(6-biotinylhexanediamin-1-yl)carboxymethyl]tropan-3-ol          diester)”           /codon_start=1          /product=“immunoglobulin light chain variable region”          /protein_id=“AAK29667.1”           /db_xref=“GI:13549148”/translation=“DIQMTQSPSTLSASVGDTVTIACRASQSINGYLAWYQQKPGKAPNLLIFKASTLQSGVPSRFSGSGYAREFTLTISSLQPDDFATYYCQQYYT HSRTFGQGTQVEITRTVAAP(SEQ ID NO: 48)” BASE COUNT  85 a  98 c  79 g  77 t ORIGIN   1gacatccaga tgacccagtc tccttccacc ctgtctgcat ctgtgggaga cacagtcacc  61atcgcttgcc gggccagtca gagtattaat ggctacttgg cctggtatca gcagaaacct 121gggaaagccc ctaacctcct gatctttaag gcatctactt tacaaagtgg ggtcccatca 181aggttcagcg gcagtggata tgcgagagaa ttcacgctca ccatcagcag cctgcagcct 241gatgattttg caacttatta ctgccaacag tattatactc actcccggac gttcggccaa 301gggacccagg tggaaatcac acgaactgtg gctgcacca (SEQ ID NO: 49)

REFERENCES FOR EXAMPLE II

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Biophys. Res. Commun. 204, 57-62-   10. Paul, S., Li, L., Kalaga, R., Wilkins-Stevens, P., Stevens, F.    J., and Solomon, A. (1995) J. Biol. Chem. 270, 15257-15261-   11. Kalaga, R., Li, L., O'Dell, J., and Paul, S. (1995) S. J.    Immunol. 155, 2695-2702-   12. Gololobov, G., Sun, M., and Paul, S. (1999) Mol. Immunol. 36,    1215-1222-   13. Liu, E., Prasad, L., Delbaere, L. T., Waygood, E. B., and    Lee, J. S. (1998) Mol. Immunol. 35, 1069-1077-   14. Baldwin, E., and Schultz, P. G. (1989) Science 245,1104-1107-   15. Gao, Q.-S., Sun, M., Rees, A., and Paul, S. (1995) J. Mol. Biol.    253, 658-664-   16. Schowen, R. L. (1988) Mechanistic principles of enzyme activity,    119-164-   17. Harel, M., Su, C. T., Frolow, F., Ashani, Y., Silman, I., and    Sussman, J. L. (1991) J. Mol. Biol. 221, 909-918-   18. Bone, R., Sampson, N. S., Bartlett, P. A., and    Agard, D. A. (1991) Biochemistry 30, 2263-2272-   19. Sampson, N. S., and Bartlett, P. A. (1991) Biochemistry 30,    2255-2263-   20. Tramontano, A., Janda, K. D., and Lemer, R. A. (1986) Proc.    Natl. Acad. Sci. USA 83, 6736-6740-   21. Charbonnier, J. B., Golinelli-Pimpaneau, B., Gigant, B.,    Tawfik, D. S., Chap, R., Schindler, D. G., Kim, S. H., Green, B. S.,    Eshhar, Z., and Knossow, M. (1997) Science 275, 1140-1142-   22. Bencsura, A., Enyedy, I., and Kovach, I. M. (1995) Biochemistry    34, 8989-8999-   23. Mody, R. K., Tramontano, A., and Paul, S. (1994) Int. J. Pept.    Prot. Res. 44, 441-447-   24. Paul, S., Sun, M., Mody, R., Eklund, S. H., Beach, C. M.,    Massey, R. J., and Hamel, F. (1991) J. Biol. Chem. 256, 16128-16134-   25. Clackson, T., Hoogenboom, H. R., Griffiths, A. D., and    Winter, G. (1991) Nature 352, 624-628-   26. McCafferty, J., Fitzgerald, K. J., Earnshaw, J., Chiswell, D.    J., Link, J., Smith, R., Kenten, J. (1994) Appl. Biochem.    Biotechnol. 47, 157-173-   27. Tyutyulkova, S., Gao, Q.-S., Thompson, A., Rennard, S., and    Paul, S. (1996) Biochimica. Biophysica. Acta. 1316, 217-223-   28. Paul, S., Sun, M., Mody, R., Tewary, H. K., Stemmer, P.,    Massey, R. J., Gianferrara, T., Mehrotra, S., Dreyer, T.,    Meldal, M. (1992) J. Biol. Chem. 267, 13142-13145-   29. Anand, N. N., Mandal, S., MacKenzie, C. R., Sadowska, J.,    Sigurskjold, B., Young, N. M., Bundle, D. R., and    Narang, S. A. (1991) J. Biol. Chem. 266, 21874-21879-   30. Tyutyulkova, S., Gao, Q.-S., and Paul, S. (1995) Antibody    Engineering Protocols 51, 377-394-   31. Sun, M., Gao, Q.-S., Kirnarskiy, L., Rees, A., and    Paul, S. (1997) J. Mol. Biol. 271, 374-385-   32. Legendre, D., Laraki, N., Graslund, T., Bjornvad, M. E.,    Bouchet, M., Nygren, P. A., Borchert, T. V., Fastrez, J. (2000) J.    Mol. Biol. 296, 87-102-   33. Wong, L., Wong, L., Radic, Z., Bruggemann, R. I., Hosea, N.,    Berman, H. A., and Taylor, P. (2000) Biochemistry 39, 5750-5757-   34. Tramontano, A. (1994) Appl. Biochem. Biotechnol. 47, 257-275-   35. Zhou, G. W., Guo, J., Huang, W. Fletterick, R. J., and    Scanlan, T. S. (1994) Science 265, 1059-1064-   36. Rahil, J., and Pratt, R. F. (1994) Biochemistry 33, 116-25-   37. Haux, J. E., Quistad, G. B., and Casida, J. E. (2000) Chem Res    Toxicol 13, 646-51-   38. Pollack, S. J., Hsiun, P., and Schultz, P. G. (1989) J. Am.    Chem. Soc. 111, 5961-5962-   39. Li, L., Sun, M., Gao, Q.-S., and Paul, S. (1996) Mol. Immunol.    33, 593-600-   40. Paul, S., Volle, D. J., Powell, M. J., and    Massey, R. J. (1990) J. Biol. Chem. 265, 11910-11913-   41. Shuster, A. M., Gololobov, G. V., Kvashuk, O. A., Bogomolova, A.    E., Smirnov, I. V., and Gabibov, A. G. (1992) Science 256, 665-667-   42. Tawfik, D. S., Chap, R., Green, B. S., Sela, M., and Eshhar,    Z.(1995) Proc. Natl. Acad. Sci. 92, 2145-2149-   43. Takahashi, N., Kakinuma, H., Hamada, K., Shimazaki, K.,    Yamasaki, Y., Matsushita, H., Nishi, Y. (2000) J Immunol Methods    235, 113-120-   44. Wirsching, P., Ashley, J. A., Lo, C. H., Janda, K. D.,    Lemer, R. A. (1995) Science 270, 1775-82-   45. Nossal, G. J. V. (1995) Annu. Rev. Immunol. 13, 1-27.

EXAMPLE III CRTAs: Covalently Reactive Transition State Analogs

The unexpected observation that an example phosphonate monoester (1)expresses covalent reactivity with serine proteases has allowed thedesign of novel CRTSAs. These compounds are mimics of the transitionstate by virtue of their tetrehedral character and the negative chargecarried on the oxygen atom. At the same time, the phosphorous atom issufficiently electrophilic to permit covalent binding to active siteserine residues. Thus, the CRTSAs combines substrate like covalentreactivity with transition state mimicry. These properties impartgreater selectivity to the CRTSAs for serine protease binding. Whencombined with appropriate flanking peptides, the CRTSAs become specificfor individual catalytic antibodies directed against different antigens.The CRTSAs of the invention may be utilized in a variety ofapplications. These include:

-   -   Selection of high turnover, specific catalytic Abs from display        libraries    -   Selective inhibition of pathogenic autoantibodies    -   Use as immunogens to stimulate the stimulate catalytic antibody        formation, followed by screening for covalent CRTSA binding to        identify the best catalysts.        CRTSA Structure: See FIGS. 16 and 17.        CRTSAs in which the covalent reactivity and transition state        mimicry are properly balanced to permit selective catalyst        binding are disclosed. Structural principles underlying CRTSA        design are:    -   The R2 group in 2 is an electron withdrawing group composed, for        example, of substituents 3-20 shown in FIG. 16. This increases        the covalent nucleophilicity of the phosphorous without        compromising the transition state character of 2. 3-20 represent        substituents with varying electron withdrawing capacity. The        ideal substituent is one that permits selective binding to the        active site of the desired catalyst without binding other        catalysts that utilize nucleophilic covalent mechanisms. For        example, increasing the covalency of the phosphorous to very        high levels is undesirable because this permits it to bind        enzymes essential to life, such as acetylcholinesterase.        Decreases in the covalency of the phosphorous are achieved using        21-37 as the R2 substituent.    -   R1 is a peptide epitope intended to permit high affinity binding        to the desired catalytic antibody. The size of this epitope is        usually 5-15 amino acids in length. The sequence of the peptide        corresponds to epitopes in any desired target of the antibodies,        e.g., beta-amyloid, IgE, Il-8, tumor necrosis factor, gp120,        EGFR and plasminogen activating inhibitor-1.    -   To further increase the specificity of CRTSAs for individual        antigen-specific catalysts, R2 is composed of 38-47, which        consist of electron withdrawing or electron donating groups        extended with a peptide epitope capable of being recognized by        the desired catalyst. Insertion of peptide sequences on both        sides of the phosphorous center is desirable to increase the        specificity of the CRTSA.        Determination of optimal CRTSA structure:        Four criteria are considered when designing the structure of the        CRTSA.    -   Potency of inhibtion of catalytic activity (Ki). The best CRTSA        is identified by screening a panel of CRTSA structures for the        ability to inhibit non-Ab serine proteases and Ab serine        proteases.    -   Irreversible CRTSA binding to non-Ab serine proteases and Ab        serine proteases under denaturing conditions        (SDS-electrophoresis; see manuscripts for method)    -   Ability of CRTSAs to select high turnover, specific catalysts        from displayed antibody libraries (see manuscripts for phage        display methods; other display methods such as bacterial and        yeast display are also suitable).    -   Immunization of experimental animals with CRTSAs followed by        analysis of polyclonal serum antibodies and monoclonal        antibodies from the immunized animals for the desired catalytic        activity.    -   FIGS. 16 and 17 depict a series of electron withdrawing or        electron donating substituents with or without flanking peptide        epitopes at position R.

-   EGFR Met-Glu-Glu-Asp-Gly-Val-Arg-Lys-Cys (SEQ ID NO: 50)    -   Cys-Glu-Gly-Pro-Cys-Arg (SEQ ID NO: 51)

-   HIVgp120 Lys-Gln-Ile-Ile-Asn-Met-Trp-Gln-Gllu-Val-Gly (SEQ ID NO:    52)    -   Ala-Met-Tyr-Ala (SEQ ID NO: 53)

-   TNF α Leu-Ala-Asn-Gly-Val-Glu-Leu (SEQ ID NO: 54)    -   Asp-Asn-Gln-Leu-Val-Val-Pro (SEQ ID NO: 55)

-   IL-1β Pro-Lys-Lys-Lys-Met-Glu-Lys (SEQ ID NO: 56)    -   Phe-Val-Phe-Asn-Lys-Ile-Glu (SEQ ID NO: 57)        Compounds I-V depicted in FIG. 18 have several utilities.

-   1. Neutral diester I and similar compounds for phage Ab selection,    inhibition of pathogenic Abs and elicitation of Ab responses in    experimental animals in instances where the reaction to be catalyzed    involves covalent binding and hydrolysis of bonds linking    non-positively charged flanking groups.

-   2. Neutral diester II and III and similar compounds with weakened    covalent reactivity for the same purposes as in 1. above. In these    compounds, substitution of one or both of the phenyl groups for    methyl groups lowers the electrophilicity of the phosphorous and    thereby imparts instability to the covalent bond between the    phosphorous and the active site nucleophiles in catalysts.

-   3. Neutral monoester IV and V for the same purposes as described in    1 above.

In another embodiment of the present invention, R1 and R2 are thepeptide epitopes corresponding to peptide determinants in the desiredtarget antigen, and R3 is an electron withdrawing or electron donatinggroup designed to increase and decrease the covalent reactivity of thephosphorus atom. For example, structures 3-20 in FIG. 16 can serve aselectron withdrawing groups, and structures 21-37 can serve as electrondonating groups. Methods for synthesis of these compounds are well-knownin the art. It is possible that placing the electron withdrawing groupR3 on the N-terminal side of the phosphonate ester leads to a change inthe leaving group upon formation of the phosphonyl-catalyst complex,i.e., liberation of the the N-terminal peptide component instead of theC-terminal component that is customarily liberated. However, for thepurposes described in this patent application, this alteration inreaction products is inconsequential, as in both cases, the covalentadduct formation will result in inhibition of antibody catalyticactivity, selective binding of catalysts for the purpose of their

isolation, and stimulation of B cell clonal selection for the purpose ofinduction of catalytic antibody synthesis. The structure of this type ofCRTSA is shown in FIG. 20.

EXAMPLE IV Passive Immunization with the Catalytic Antibodies of thePresent Invention

There are many areas in medicine where monoclonal antibodyadministration is providing clinical benefit. In the field of organtransplantation, a MoAb (OKT3) which binds to the T cell receptor hasbeen employed to deplete T cells in vivo. Additionally, MoAbs are beingused to treate graft v. host disease with some success. A clinical trialhas been established which is assessing the ability of anti-CD4 moAB todeplete a subset of T cells in the treatment of multiple schlerosis.Accordingly, methods of administration of monoclonal antibodies are wellknown to clinicians of ordinary skill in the art. An exemplary methodand dosage schedule are provided in a phase III, randomized, controlledstudy of chemotherapy alone or in combination with a recombinant moAB tothe oncogene HER2.

All patients randomized to the recombinant humanized MoAb Her2 arm ofthe study will receive treatment as a 4 mg/lkg I.V. loading dose on Day0 (the first day of the MoAb HER2 infusion, or the day of randomizationfor patients in the control group), then weekly as a dose of 2 mg/kgI.V. through out the course of the study. All patients will be monitoredduring each study visit by a clinical assessment, a symptom directedphysical examination (if appropriate) and laboratory tests. Routinetumor evaluations will be conducted for all patients at prescribedintervals during the study. All adverse events will be recorded.

The administration of the catalytic antibodies of the present inventionwill be done as described above for the HER2 monoclonal antibody. As inthe HER2 study, following infusion, patients will be assessed todetermine the efficacy of the administered catalytic antibody.

Should the catalytic antibodies administered as above give rise toundesirable side effects in the patient, the immunizing CRTSAs will beadministered to covalently inhibit the action of the catalyticantibodies.

EXAMPLE V Active Immunization Using the CRTSAs of the Present Invention

Active immunization will be done using previously developed methods withvaccines designed to elicit protective antibody responses against thedesired antigens [82, 83]. For example, the CRTSAs mixed with a suitableadjuvant formulation such as alum can be admimistered intramuscularly ata dose optimized for maximum antibody synthesis (100-1000 μg/kg bodyweight), and two or three booster injectijns can be administed at 4 weekintervals, until the catalytic antibody concentration in the serumreaches plateau levels. The protective immunity so generated isanticipated to last for several years, because vaccination will resultin formation of specific, long lived memory cells that can be stimulatedto produce antibodies upon exposure to the offending organism or cancercell. Descriptions and methods to determine the catalytic antibodyconcentrations are set forth in Examples I and II. Because antibodysynthetic response to most antigens are T cell dependent, an appropriateT cell epitope can be incorporated into the immunogen by peptidesynthesis. Alternatively, a carrier such as keyhole limpet hemocyanincan be conjugated to the CRTSA via coupling through lys side chain aminogroups or Cys side chain sulfahydryl groups to maximize the antibodyresponse if necessary.

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

1. A covalently reactive transition state antigen analog (CRTSA),comprising the following structural formula:R₁-E-R₂ wherein R₁ is a peptide sequence of an epitope of a targetprotein antigen, E is an electrophilic covalently reactive centerbearing a partial or full negative charge and R₂ is an electronwithdrawing or electron donating substituent, R₂ optionally furthercomprising a flanking peptide sequence, further comprising Y between R₁and E, wherein Y is a positively charged amino acid adjacent to theelectrophilic center selected from the group consisting of lysine,arginine or analogs thereof.
 2. The CRTSA as claimed in claim 1, whereinsaid electrophilic reaction center is selected from the group consistingof a phosphonate moiety, a boronate moiety and a vanadate moiety.
 3. TheCRTSA as claimed in claim 1, wherein Y is an amino acid analog furthercomprising an electron withdrawing or electron donating substituent. 4.The CRTSA of claim 1 wherein Y is an electron withdrawing substituentselected from the group consisting of structures 3-20 of FIG. 16 andstructures 38-47 of FIG.
 17. 5. The CRTSA of claim 1 wherein Y is anelectron donating substituent selected from the group consisting ofstructures 21-37 of FIG.
 17. 6. The CRTSA as claimed in claim 1, whereinsaid epitope of a target protein antigen is an epitope present inproteins selected from the group consisting of tumor necrosis factor,epidermal growth factor recepor, interleukin-1, gp120, gp160, gag, pol,hepatitis B surface antigen, bacterial exotoxins, EGF, TGFα, p53,prostate specific antigen, carcinoembryonic antigen, prolactin, humanchorionic gonadotropin, c-myc, c-fos, c-jun, epidermal growth factorreceptor, HER-2, prolactin receptors, steroid receptors, and IL-4. 7.The CRTSA as claimed in claim 1, said CRTSA elicits catalytic antibodyproduction to epidermal growth factor receptor, wherein R₁ isMet-Glu-Glu-Asp-Gly-Val-Arg-Lys-Cys (SEQ ID NO: 50); Y is Lys or ananalog thereof; E is a phosphonate monoester; and R₂ comprises theflanking sequence Cys-Glu-Gly-Pro-Cys-Arg (SEQ ID NO: 51).
 8. The CRTSAas claimed in claim 1, said CRTSA elicits catalytic antibody productionto gp120, wherein R₁ is Lys-Gln-Ile-Ile-Asn-Met-Trp-Gln-Glu-Val-Gly (SEQID NO: 52); Y is Lys or an analog thereof; E is a phosphonate monoester;and R₂ comprises the flanking sequence Ala-Met-Tyr-Ala (SEQ ID NO: 53).9. The CRTSA as claimed in claim 1, said CRTSA elicits catalyticantibody production to TNFα, wherein R₁ is Leu-Ala-Asn-Gly-Val-Glu-Leu(SEQ ID NO: 54); Y is Lys or an analog thereof; E is a phosphonatemonoester; and R₂ comprises the flanking sequenceAsp-Asn-Gln-Leu-Val-Val-Pro (SEQ ID NO: 55).
 10. The CRTSA as claimed inclaim 1, said CRTSA elicits catalytic antibody production to IL-1β,wherein R1 is Pro-Lys-Lys-Lys-Met-Glu-Lys (SEQ ID NO: 56); Y is Lys oran analog thereof; E is a phosphonate monoester; and R₂ comprising thesequence Phe-Val-Phe-Asn-Lys-Ile-Glu (SEQ ID NO: 57).